Characterization of new putative leaf growth regulators...

Characterization of new putative leaf growth

regulators involved in organelle gene expression

Pieter HEEREMANS

Master’s dissertation submitted to obtain the degree of

Master of Science in Biochemistry and Biotechnology

Major Plant Biotechnology

Academic year 2014-2015

Promoters: Prof. Dr. Dirk Inzé and Dr. Nathalie Gonzalez

Scientific supervisor: Jonas Blomme

UGent - Department Plant Biotechnology and Bioinformatics

VIB - Department Plant Systems Biology

Research Group - Systems Biology of Yield

1

Acknowledgements

First of all I would like to thank Jonas Blomme for his excellent guidance and support even

from the other side of the world. It has been a very educational year and it has fueled my

interest in science.

I would also like to express my gratitude to Prof. Dr. Dirk Inzé for giving me the opportunity to

do my Master Dissertation in the Systems Biology of Yield lab.

I really appreciated the nice atmosphere and people in the lab, it made the last few months

feel like a breeze. Special thanks go out to Lisa Van Den Broeck, Mattias Vermeersch,

Alexandra Baekelandt and Marieke Dubois for their scientific advice.

I am also extremely grateful to Dr. Nathalie Gonzalez for her expertise, valuable guidance and

support and especially for taking me under her wing while Jonas was abroad.

I am also thankful to my best friends Hannes De Brouwer and Arian Zand and my parents and

sister for their unceasing encouragement and support. I couldn’t wish for better friends or

family.

2

Table of contents

Acknowledgements ................................................................................................................................. 1

Table of contents ..................................................................................................................................... 2

List of abbreviations ................................................................................................................................ 5

List of figures ........................................................................................................................................... 6

List of tables ............................................................................................................................................ 6

Summary ................................................................................................................................................. 7

Samenvatting ........................................................................................................................................... 8

Part 1: Introduction ................................................................................................................................. 9

1. A changing world and the need for crop improvement ...................................................................... 9

2. Ways to improve crop yield ............................................................................................................... 10

3. The Leaf development regulatory network ....................................................................................... 11

4. Organelles as a source of leaf growth regulators .............................................................................. 14

4.1.The importance of organelle function in leaf development ....................................................... 14

4.2. Gene expression in organelles.................................................................................................... 16

4.3. SWIB-family domain proteins as potential new regulators of gene expression in mitochondria

and chloroplasts ................................................................................................................................ 17

Part 2: Aim of the research ................................................................................................................... 17

1. Work package one ......................................................................................................................... 18

2. Work package two ......................................................................................................................... 19

3. Work package three ...................................................................................................................... 19

Part 3: Results ........................................................................................................................................ 20

1. Phenotypic characterization of SWIB-family domain proteins ......................................................... 20

1.1. Gene expression analysis of T-DNA insertion mutants .............................................................. 20

1.2. Leaf size analysis of T-DNA insertion mutants ........................................................................... 23

1.3. Detailed characterization of selected T-DNA insertion mutants ............................................... 25

3

1.3.1. Cellular analysis ................................................................................................................... 26

1.3.2. Root analysis ........................................................................................................................ 29

1.3.3. Ploidy analysis ..................................................................................................................... 30

1.4. Phenotypic analysis of overexpression lines .............................................................................. 33

2. SWIB-domain family proteins under stress conditions ..................................................................... 34

2.1. Gene expression analysis of a wild type line on stress .............................................................. 34

2.2. Leaf size analysis of selected T-DNA insertion mutants ............................................................. 37

3. Expression analysis of SWIB5 ............................................................................................................ 40

4. Localization analysis of SWIB-family domain proteins ...................................................................... 41

5. Interaction analysis of SWIB5 ............................................................................................................ 44

Part 4: Discussion .................................................................................................................................. 46

1. Aim of the research project ............................................................................................................... 46

2. Localization analysis of SWIB-family domain proteins ...................................................................... 46

3. Phenotypic characterization of SWIB-family domain proteins ......................................................... 47


3.2. Detailed characterization of selected T-DNA insertion mutants ............................................... 48

4. Gene expression analysis of SWIB-family domain proteins .............................................................. 49

5. SWIB-family domain proteins under stress conditions ..................................................................... 50

5.1. Gene expression analysis of a wild type line on stress .............................................................. 50


6. Interaction analysis of SWIB5 ............................................................................................................ 52

7. Conclusion ......................................................................................................................................... 53

Deel 4: Discussie .................................................................................................................................... 52

Part 5: Material and methods ............................................................................................................... 57

1. Material ............................................................................................................................................. 57

1.1. Plant material ............................................................................................................................. 57

1.2. Growth media ............................................................................................................................. 58

4

2. Methods ............................................................................................................................................ 59

2.1. General methods ........................................................................................................................ 59

2.2. Gene expression analysis of T-DNA insertion mutants .............................................................. 60

2.3. Gene expression analysis under stress conditions ..................................................................... 61

2.4. Ploidy analysis of T-DNA insertion mutants ............................................................................... 62

2.6. Localization analysis of the SWIB-family domain proteins ........................................................ 65

2.7. Interaction analysis of SWIB5 ..................................................................................................... 66

2.8. Leaf size analysis ......................................................................................................................... 67

2.9. Cellular analysis .......................................................................................................................... 68

2.10. Root analysis ............................................................................................................................. 69

References ............................................................................................................................................. 70

Attachments .......................................................................................................................................... 74

5

List of abbreviations

ATP Adenosine triphosphate

DNA Deoxyribonucleic acid

GFP Green fluorescent protein

GM Genetic modification

GOI Gene of interest

GOF Gain of function line

GS Genomic selection

GUS β-glucuronidase

INT Interacting partner of SWIB5

LOF Loss-of-function line

LRD Lateral root density

LRD Lateral root density

MAS Marker assisted selection

NAP Nucleoid associated protein

NF Norflurazon

OE Overexpression line

PGD Plant genetic diversity

PPR Pentatricopeptide repeat

PRL Primary root length

QC Quiescent center

QTL Quantitative trait locus

RNA Ribonucleic acid

ROS Reactive oxygen species

SAM Shoot apical meristem

SWIB SWI/SNF complex B

T1-2-3 First-second-third transgenic generation

TAIR The Arabidopsis Information resource database

TAP Tandem affinity purification

T-DNA Transfer-DNA

WP Work package

WT Wild type

6

List of figures Figure 1: Leaf development in Arabidopsis thaliana.

Figure 2: The Arabidopsis thaliana crumpled leaf (crl) mutant.

Figure 3: The effect of norflurazon (NF) on Arabidopsis thaliana seedlings.

Figure 4: The Arabidopsis thaliana tang2 and otp439 mutants.

Figure 5: Relative expression levels of SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 and INT90 in their T-DNA insertion lines.

Figure 6: Differences in leaf size for all leaves in the different T-DNA insertion mutants compared to the wild type.

Figure 7: The difference in leaf size between four selected mutants and the wild type.

Figure 8: Leaf size, leaf cell number and average pavement cell size of the third leaf of the T-DNA insertion mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared to the wild type.

Figure 9: Lateral root length (LRL), primary root length (PRL) and lateral root density (LRD) of the T-DNA insertion mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared to the wild type.

Figure 10: Ploidy level of the T-DNA insertion mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared to the wild type.

Figure 11: Overexpression lines for the SWIB1 gene.

Figure 12: Overexpression lines for the SWIB6 gene.

Figure 13: Relative expression of the SWIB1, SWIB2, SWIB3, SWIB4, SWIB5, SWIB6, INT80 and INT90 genes on different stress-inducing media.

Figure 14: The ratio of the leaf size on stress-inducing media to the leaf size on control medium.

Figure 15: Differences in leaf size for all leaves in the selected T-DNA insertion lines for SWIB3, SWIB4, SWIB6 and INT80, compared to the wild type on different stress-inducing media containing 25 mM mannitol, 50 mM NaCl, 10 µM rotenone, 1 mM H2O2 or 50 µM antimycin A.

Figure 16: pSWIB5::GFP::GUS expression in the 5, 9 and 12 day old seedlings.

Figure 17: CLSM image of Nicotiana benthamiana leaves after co-infiltration of the p35S::GENE::GSGreen construct with the mitochondrion or the chloroplast marker.

List of tables Table 1: Overview of the T-DNA insertion lines used in the expression analysis and the leaf size analysis.

Table 2: The proteins found in the tandem affinity purification experiment using p35S::SWIB5::GSGreen seedlings.

Table 3: Overview of the T-DNA insertion lines for the genes of interest.

Table 4: Overview of the codes used for the lines containing the p35S::GENE constructs.

Table 5: Overview of the codes used for the lines containing the pSWIB5::GFP::GUS constructs.

7

English summary

In order to increase crop yield we need to obtain a better understanding of all aspects of plant

development and this includes the influence of organelles. The majority of the proteins that

are active in mitochondria and chloroplasts are encoded in the nucleus. The nuclear-encoded

SWI/SNF complex B (SWIB) domain proteins are potential new regulators of gene expression

in these organelles. In silico searches showed that in Arabidopsis thaliana there are six

proteins of low molecular mass that contain a SWIB domain. SWIB1 is targeted to the

cytoplasm. SWIB2, SWIB3 and SWIB4 are targeted to the chloroplasts, SWIB4 is also targeted

to the nucleus, SWIB5 to the mitochondria and SWIB6 to both mitochondria and chloroplasts.

In Arabidopsis thaliana, SWIB4 was shown to associate with the nucleoid of the chloroplasts

and by expressing it in Escherichia coli it was shown to be involved in nucleoid condensation.

In Arabidopsis thaliana, SWIB5 loss- and gain of function lines show defects in leaf

development. In this research project we aim to characterize different SWIB-family domain

proteins and two interacting partners of SWIB5 both phenotypically and at the molecular

level.

We observed changes in leaf size in loss- and gain-of-function lines for the different SWIBs.

For example, in a swib3 mutant we found a strong decrease in leaf size whereas swib4 and

swib6 mutants produced larger leaves. SWIB3, SWIB4, SWIB6 and INT80 might play a role in

cell cycle regulation as mutants of these genes often showed defects in cell proliferation. It

was also shown that SWIB5 is expressed in cells and tissues that are proliferating and actively

expanding such as cells of the root tip or at the edges of developing leaves. We also found that

the expression of the SWIB genes are highly responsive to mild and strong stress conditions in

mitochondria and chloroplasts. Overall, we observed a downregulation of the SWIB genes as

a response to these stress conditions. Also, the loss-of-function lines for the different SWIBs

showed an increased sensitivity to these stress conditions compared to the wild type.

8

Nederlandse samenvatting

Om de opbrengst van gewassen te kunnen verhogen, gaan we onze kennis over alle aspecten

van plantontwikkeling moeten uitbreiden. Een van deze aspecten is de invloed van

celorganellen op plantontwikkeling. Het grootste deel van de proteïnen die actief zijn in de

mitochondriën en chloroplasten worden aangemaakt in de nucleus. SWI/SNF complex B

(SWIB) domein proteïnen worden aangemaakt in de nucleus en spelen mogelijks een rol in de

regulatie van genexpressie in mitochondriën en chloroplasten. Het werd reeds aangetoond

dat er in Arabidopsis thaliana zes proteïnen worden aangemaakt die een SWIB domein

bevatten. SWIB1 wordt naar het cytoplasma gestuurd. SWIB2, SWIB3 en SWIB4 gaan naar de

chloroplasten, SWIB4 gaat ook naar de nucleus, SWIB5 naar de mitochondriën en SWIB6 naar

zowel de mitochondriën als de chloroplasten. In Arabidopsis thaliana werd reeds aangetoond

dat SWIB4 associeert met het chloroplast nucleoid en in Escherichia coli werd aangetoond dat

SWIB4 een rol speelt in condensatie van het nucleoid. Het werd aangetoond dat SWIB5 gain-

of-function en loss-of-function lijnen defecten vertonen in bladontwikkeling en naar

aanleiding van deze bevindingen is het ons doel om de andere SWIBs ook fenotypisch te

karakteriseren. Daarnaast willen we ook de stress respons van de SWIBs bestuderen.

We hebben aangetoond dat er een effect was op de bladgrootte in loss- en gain of function

lijnen voor de verschillende SWIBs. De swib3 mutant bijvoorbeeld produceerde bladeren die

veel kleiner waren in vergelijking met het wild type. De swib4 en swib6 mutanten daarentegen

produceerden grotere bladeren. In loss-of-function lijnen voor de SWIB3, SWIB4, SWIB6 en

INT80 genen was het effect op bladgrootte vooral te wijten aan defecten in celdeling en

daarom vermoeden we dat deze genen een rol spelen in de regulatie van de cell cyclus. We

toonden ook aan dat SWIB5 tot expressie komt in cellen en weefsels die actief aan het delen

zijn zoals bijvoorbeeld de worteltip of de randen van ontwikkelende bladeren. De SWIB genen

in wild type planten werden meestal neergereguleerd wanneer de planten werden

getransfereerd naar stress-inducerende media. Daarnaast werden vier T-DNA insertie

mutanten geselecteerd, gegroeid op stress-inducerende media en hun bladgrootte werd

bepaald. Dit toonde aan dat deze mutanten veel gevoeliger waren aan stress in de

mitochondriën of in de chloroplasten in vergelijking met het wild type.

Part 1: Introduction

9


1. A changing world and the need for crop improvement

Recently it has become clear that in order to keep feeding the rapidly growing human

population, the production of food will have to increase and this will have to be combined

with a reduction in the use of fertilizers (Edgerton, 2009). However, it will be very challenging

to accomplish this and there are even concerns about sustaining crop yield and quality since

new environmental and biotic threats are emerging. Furthermore, in the past few years an

increasing amount of farmable land has been used to grow crops for the production of

biofuels, therefore adding even more pressure on the food supply (Foley et al, 2005).

We will not only need more food for our growing human population, but the quality of the

food will need to be improved as well (Edgerton, 2009). One way to achieve this is to increase

the micronutrient content. Micronutrients have to be obtained through the diet as humans

do not produce them. Staple crops are our main food source but they are generally deficient

in one or the other micronutrient (Beyer, 2010). Recently, it has become possible to produce

a number of micronutrients in crops as the genes that are involved in the corresponding

biosynthesis pathways were identified. For example, the endosperm of rice grains lacks

several essential micronutrients, such as provitamin A. This leads to vitamin A deficiency in

people that predominantly eat rice. As a result, vitamin A deficiency is problematic in at least

26 countries, including highly populated areas of Asia, Africa, and Latin America. This problem

was addressed by engineering the provitamin A biosynthetic pathway into rice (Oryza sativa)

endosperm (Ye et al, 2000).

In addition to increasing crop yield and quality, the use of chemical fertilizers will have to be

reduced as well in the future. (Rogers & Oldroyd, 2014). Nitrogen is abundant in the

atmosphere but it cannot be directly assimilated by plants. Therefore, chemical fertilizers are

often used to increase the yield of crops. The problem with the application of chemical

fertilizers is that their production requires a lot of energy leading to greenhouse gas emissions.

This problem could be addressed using synthetic biology for example by engineering the

nitrogenase enzyme present in nitrogen-fixing bacteria in crops (Liu & Stewart, 2015).

Although challenging, it is a viable long-term goal.


10

The traits that are selected in breeding programs are predominantly increasing crop yield

under normal conditions. This selection strategy will result in crops with a higher yield under

normal conditions but it is possible that they are also more sensitive to stress. Therefore it is

better to use different selection strategies that also include stress conditions (Marshall et al,

2012).

2. Ways to improve crop yield

There are different ways to increase the yield of crops. The first option is to increase the area

of farmable land. The consequence of this option is that existing ecosystems would be

disrupted. Therefore a second option, improving the productivity of the already existing

farmland through crop improvement, is highly preferred (Edgerton, 2009).

Breeding programs are an effective approach to improve crop yield and quality. (Tester &

Langridge, 2010). Plant breeders predominantly focus on traits that have a high potential to

improve the crop such as drought tolerance traits. To increase the efficiency of breeding

programs, new technologies based on improvements in phenotypic and genotypic analyses

are constantly being developed to select for desirable traits (Ray & Satya, 2014).

A method that is often used to create varieties with improved traits is marker assisted

selection (MAS) (Tester & Langridge, 2010). This breeding approach uses a marker such as a

specific phenotype, a chemical tag, a particular DNA or RNA motif or chromosomal segment

that associates with a desirable trait. A DNA marker closely linked to a certain locus with a

phenotype can be used to predict if a plant will display that phenotype.

Gene pyramiding is a breeding approach based on MAS used to create durable disease

resistances by using more than one resistance gene against a certain pathogen (Tester &

Langridge, 2010). Genomic selection (GS) is also based on MAS and in this breeding approach

genetic markers covering the whole genome are used to select all desirable quantitative trait

loci (QTLs) (Tester & Langridge, 2010). QTLs are stretches of DNA linked to, or containing the

genes that underlie a quantitative trait. The large number of single nucleotide polymorphisms

(SNP) discovered by genome sequencing makes GS an interesting approach to incorporate


11

desirable alleles at many loci that only have a small effect when used individually (Tester &

Langridge, 2010).

Crop improvement will also benefit from an increase in the available plant genetic diversity

(PGD) (Govindaraj et al, 2015). Therefore, it is important to preserve the available PGD and to

discover new sources of PGD such as landraces and wild relatives of crop species.

Genetic modification (GM) is also considered as a great tool for crop improvement as it allows

the generation of transgenic plants (Szabala et al, 2014). Transgenic plants have one of their

genes misexpressed or contain at least one gene that is derived from another species. An

example of GM in crops are transgenic maize plants expressing the cry genes derived from

Bacillus thuringiensis (Bt), thereby allowing them to produce pesticides (Bt toxins) (Koch et al,

2015; Tian et al, 1991). The production of transgenic crops through GM and their evaluation

is an active area of research, but the access of farmers to transgenic crops is restricted in a lot

of countries because of political and ethical issues.

3. The Leaf development regulatory network

To progressively increase the yield of crops we will need to get a better understanding of how

plant growth is regulated. The Systems Biology of Yield Group focuses on the processes that

control leaf development as a model for plant growth. The group is interested in leaf

development because leaves are the main site of photosynthesis in plants and therefore crop

yield is strongly dependent on leaf size and shape.

Leaves originate from the shoot apical meristem (SAM). In Arabidopsis thaliana, at the

periphery of the SAM, groups of cells form primordia which grow out as rod-shaped

protrusions. The development of primordia into mature leaves can be divided into two

partially overlapping phases (Gonzalez et al, 2012). During the first phase, the proliferation

phase, new small cells of relatively constant size are generated through cell growth followed

by mitotic division. This process takes place throughout the entire primordium. In the second

phase, the cell expansion phase, the cells are growing through turgor-driven expansion and

they start to differentiate. During the transition phase between cell proliferation and


12

expansion, the cells start to differentiate along a basipetal gradient (from leaf tip to base)

(figure 1).

The regulation of cell proliferation and expansion is strongly connected. For example, it was

shown that a decrease in leaf size caused by a defect in cell proliferation can be (partly)

compensated by cell expansion (Hisanaga et al, 2015).

Figure 1: Leaf development in Arabidopsis thaliana. The first phase of leaf development is called the

proliferation phase and is characterized by the generation of new small cells of relative constant size. The second

phase is called the expansion phase and is characterized by turgor-driven cell expansion and differentiation.

During the transition phase, the cells start to differentiate along a basipetal gradient (from leaf tip to base). The

red arrow indicates the amount of cells that were recruited to the primordium and the black arrow indicates the

start of the transition phase.

In many plant species, including Arabidopsis thaliana there is a shift from a mitotic cell cycle

to an endoreduplication cycle in the leaf during the transition phase (De Veylder et al, 2011).

During the endoreduplication cycle, the nuclear DNA is replicated without mitotic cell division,

resulting in an increase in the ploidy level. The ploidy level is often correlated with level of cell

differentiation and cell size. It is also often inversely correlated with genome size and this has

led to the hypothesis that a higher ploidy level was used to compensate for a smaller genome

size (Kondorosi et al, 2000).

The final leaf size is affected by different processes that control leaf development: the

recruitment of cells to the primordium, cell division and expansion and cell differentiation. By

making changes in one or more of these processes it is possible to influence the final leaf size.

Many genes that are important in the regulation of these processes have already been

described.


13

The SAM cells maintain a pluripotent stem cell population and are responsible for producing

leaves or flowers. The size of the SAM can be of influence to final leaf size. SAMBA is a negative

regulator of the APC/C complex which initiates the transition from metaphase to anaphase.

The Arabidopsis thaliana samba mutant has a larger SAM and produces larger seeds, leaves

and roots. (Eloy et al, 2012).

The amount of cells that are recruited to the primordium can also be of influence to final leaf

size. The Arabidopsis thaliana struwwelpeter (swp) mutant for example shows a decrease in

the amount of cells recruited to the primordium and produces smaller leaves which is caused

by a decrease in the leaf cell number (Autran et al, 2002).

The time it takes to complete the cell cycle is of influence to the leaf cell number and thereby

also to the final leaf size. One of the most crucial checkpoints during the cell cycle is the exit

from mitosis which is controlled by the Anaphase-Promoting Complex/Cyclosome (APC/C).

APC10 is a subunit of the APC/C complex and in Arabidopsis thaliana, overexpression of APC10

produces larger leaves which is caused by an increase in the leaf cell number (Eloy et al, 2011).

Cell expansion is also of influence to final leaf size. EXPANSINs are proteins that are associated

with the cell wall and are involved in cell wall loosening and extension of the cell wall. In

Arabidopsis thaliana, overexpression of EXP10 resulted in the production of larger leaves

which was caused by an increase in cell size.

During leaf development, dispersed meristematic cells (DMCs) in the epidermis differentiate

into specific cell types such as guard cells and pavement cells. A difference in the amount of

DMCs can be of influence to final leaf size. The Arabidopsis thaliana peapod mutant shows an

increased amount of DMCs and produces dome-shaped leaves with an increased lamina size

(White, 2006).

Through reverse and forward genetics, numerous genes have been described that are involved

in the regulation of cell proliferation and/or expansion. The large and increasing amount of

genes involved in leaf development illustrates the complexity of the regulatory network

controlling it (Gonzalez & Inze, 2015).


14

4. Organelles as a source of leaf growth regulators

4.1. The importance of organelle function in leaf development

Mitochondria and plastids are endosymbiotic organelles that have contributed to the

evolution of present-day eukaryotic cells. According to the endosymbiotic theory (Witzany,

2006), mitochondria evolved from Proteobacteria and plastids from Cyanobacteria.

Mitochondria are present in all eukaryotic cell types and are involved in essential metabolic

and cellular processes such as respiratory ATP production, signaling, cellular differentiation

and cell death. Plastids are present in plants and algae and in plants they differentiate into

multiple subtypes depending on the cell type. Chloroplasts are one such subtype and they are

involved in essential metabolic and cellular processes such as photosynthesis and amino acid

and lipid synthesis.

The importance of plastid function in leaf development is illustrated by the Arabidopsis

thaliana crumpled leaf (crl) mutant (Hudik et al, 2014). The crl mutant is deficient for a protein

that is targeted to the plastid envelope. In the crl mutant, defects in cell cycle regulation lead

to the production of smaller leaves which is caused by the premature differentiation of DMCs

(figure 2).

Figure 2: The Arabidopsis thaliana crumpled leaf (crl) mutant. Defects in cell cycle regulation lead to the

production of smaller leaves caused by the premature differentiation of DMCs in the crl mutant (Hudik et al,

2014).


15

The importance of plastid function in leaf development is also illustrated by the effect of

norflurazon (NF), a chemical inhibitor of retrograde signaling in plastids, on seedlings (figure

3). In retrograde signaling, plastid-encoded proteins are targeted to the nucleus where they

regulate gene expression. The leaves of seedlings in which the SAM was treated with NF have

defects in the transition phase between cell proliferation and expansion, therefore showing

that proteins encoded in the plastids are important in leaf development (Andriankaja et al,

2012).

Figure 3: The effect of norflurazon (NF) on Arabidopsis thaliana seedlings. The leaves of seedlings in which the

SAM was treated with NF have defects in the transition phase between cell proliferation and expansion

(Andriankaja et al, 2012).

The importance of mitochondrion function in leaf development is illustrated by the

characterization of two pentatricopeptide repeat (PPR) proteins (TANG2 and OTP439) which

are involved in the correct splicing of NAD5 in mitochondria (Colas des Francs-Small et al,

2014). The NAD5 gene encodes a subunit of Complex I in the electron transport chain of

mitochondria. It is fragmented in five exons, belonging to three distinct transcription units. In

Arabidopsis thaliana, incorrect splicing of NAD5 caused by a defect in one of the two PPR

proteins leads to the production of smaller leaves (figure 4).


16

Figure 4: The Arabidopsis thaliana tang2 and otp439 mutants. Incorrect splicing of the NAD5 gene caused by a

defect in one of the two PPR proteins leads to the production of smaller leaves (Colas des Francs-Small et al,

2014).

4.2. Gene expression in organelles

During the evolution of endosymbiotic bacteria to mitochondria and plastids, most of the

genetic information present in the endosymbiotic bacteria was transferred to the host cell

nucleus (Liao et al, 2015). Therefore, most of the proteins present in the mitochondria and

chloroplasts in eukaryotic cells are encoded by the nuclear genome, synthesized on cytosolic

ribosomes and targeted to these organelles. The proteins that are encoded by the

mitochondria or chloroplasts themselves are often involved in essential metabolic and cellular

processes such as respiratory ATP production or photosynthesis. The regulation of the genes

that encode these proteins relies heavily on proteins encoded in the nucleus (Liao et al, 2015).

In chloroplasts for example, the majority of genes are clustered in operons and both nuclear-

encoded and chloroplast-encoded RNA polymerases are involved in the transcription of

chloroplast genes (Barkan, 2011). Also, the primary transcripts in the chloroplasts have to go

through posttranscriptional processing and this relies heavily on nuclear-encoded enzymes.

These posttranscriptional processes include endonucleolytic cleavage of di- or polycistronic

transcripts, intron splicing, 5′ and 3′ end processing and RNA editing (Liao et al, 2015).

In contrast to chloroplasts, the mitochondria do not encode their own RNA polymerases. The

transcription of genes in the mitochondria is controlled completely by the nuclear-encoded

RNA polymerases (Hedtke et al, 1999).


17

4.3. SWIB-family domain proteins as potential new regulators of gene

expression in mitochondria and chloroplasts

The SWI/SNF complex domain was first identified in Saccharomyces cerevisiae. It facilitates

transcriptional activation by chromatin remodeling. Two forms of the complex exist (complex

A and complex B) and the SWI/SNF complex B (SWIB) was found in Arabidopsis thaliana and

Chlamydophila trachomatis (Bennett-Lovsey et al, 2002).

In silico searches showed that in Arabidopsis thaliana there are six proteins of low molecular

mass that contain a SWIB domain (Melonek et al, 2012). The SWIB-family domain proteins are

targeted to the mitochondria and/or the chloroplasts (Melonek et al, 2012). SWIB2, SWIB3

and SWIB4 are targeted to the chloroplasts where they might associate with the nucleoid,

SWIB4 is also targeted to the nucleus, SWIB5 to the mitochondria and SWIB6 to both the

mitochondria and chloroplasts. SWIB1 is the only one of these six SWIB domain proteins that

is targeted to the cytoplasm.

The N-terminal sequence of SWIB4 showed high similarity to the sequence of the histone-H1-

like protein present in Chlamydophila trachomatis. This bacterium encodes the only two

SWIB-family domain proteins that are described in prokaryotes (Melonek et al, 2012). It was

shown that this histone-H1-like protein functions in chromatin remodeling during the life cycle

of the bacterium (Bennett-Lovsey et al, 2002). In Arabidopsis thaliana, SWIB4 was shown to

associate with the nucleoid of the chloroplasts. When SWIB4 was expressed ectopically in

Escherichia coli it was shown to be involved in nucleoid condensation.

The SWIB-family domain proteins also show high similarity to the nucleoid-associated proteins

(NAPs) present in bacteria. They share a low molecular mass, a high Lys content and bind both

nonspecifically to DNA. It was shown that NAPs function in chromatin remodeling during the

bacterial life cycle (Melonek et al, 2012). Because of their function in chromatin remodeling,

their association with the nucleoid and their localization, it is highly likely that these SWIB-

family domain proteins are involved in the regulation of gene expression in mitochondria

and/or chloroplasts. It was already shown that SWIB5 gain-of-function and loss-of-function

lines produced smaller leaves caused by a defect in cell proliferation. We aim to characterize

the other SWIBs as well in order to gain a better understanding of their role in leaf

development and their response to stress conditions.

Part 2: Aim of the research

18

Part 2: Aim of the research project

It was already shown that organelle functionality is of influence to plant development (Colas

des Francs-Small et al, 2014; Hudik et al, 2014). In this research project, we studied the

influence of SWIB-family domain proteins on plant development. These nuclear-encoded

proteins are targeted to the mitochondria and/or the plastids and it is possible that they are

involved in organelle gene expression regulation. The aim of the research project was to

characterize the SWIBs phenotypically and at the transcription level under both normal and

stress conditions.

We studied the six different members of the SWIB-family domain proteins (SWIB1, SWIB2,

SWIB3, SWIB4, SWIB5 and SWIB6) and two interacting partners of SWIB5 (INT80 and INT90).

The characterization of these genes was divided into three work packages (WP). In WP1 loss-

and gain-of-function lines were characterized phenotypically. In WP2, we evaluated the

response of these genes to stress-inducing compounds. Also, loss-of-function lines under

stress conditions were characterized phenotypically. In WP3 the localization of the proteins of

interest was studied. We also looked for new interacting partners of SWIB5.

1. Work package one

In the first WP of this research we characterized different loss- and gain-of-function lines

phenotypically. The expression levels of the genes of interest in their respective T-DNA

insertion lines were first evaluated using qRT-PCR. Then, these T-DNA insertion lines were

characterized in a leaf size analysis.

Four T-DNA insertion mutants for which a difference in leaf size was observed were selected

for further phenotypic characterization. First, the cell number and the average cell size of the

leaves were compared to the wild type. This allowed us to determine which of the

developmental processes were affected (cell proliferation and/or expansion) in these

mutants. Then, the primary root length, the lateral root length and the lateral root density of

the T-DNA insertion mutants was compared to the wild type. This allowed us to determine if

Part 2: Aim of the research

19

the mutants showed a root phenotype in addition to the shoot phenotype. Finally, the ploidy

level was compared to the wild type.

2. Work package two

In the second WP of this research project we evaluated the expression of the genes of interest

under stress conditions. To do this we transferred wild type seedlings from control medium

to different stress-inducing media for 24 hours and measured the expression levels of the

genes of interest. Different compound inducing osmotic stress, oxidative stress, salt stress and

stress in mitochondria and chloroplasts were used in both mild and strong concentrations. If

a gene showed a difference in expression level on medium containing a stress-inducing

compound compared to control medium, it could be involved in the stress response to that

compound.

The four selected mutants were also grown on the stress-inducing media mentioned above.

This allowed us to determine if these lines are more sensitive or tolerant to certain stress-

inducing compounds compared to the wild type.

3. Work package three

In the third WP of this research we evaluated the localization of the proteins of interest and

the expression pattern of SWIB5. The expression pattern of SWIB5 was described using a

pSWIB5::GFP::GUS line, which allowed us to determine in which cell types or tissues SWIB5 is

expressed. We evaluated the localization of SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 and

INT90 by using a p35S::GENE::GSGreen construct that was transiently expressed in Nicotiana

benthamiana in a co-localization experiment. This allowed us to determine if these proteins

are targeted to the mitochondria, the chloroplasts or another cell compartment.

To find new interacting partners of SWIB5, we performed a tandem affinity purification (TAP)

experiment using in planta material. The INT80 and INT90 interacting partners of SWIB5 were

found in a previous TAP experiment using a cell culture and to confirm the interaction of

SWIB5 with these proteins, a yeast two-hybrid experiment was performed.

Part 3: Results

20

Part 3: Results

1. Phenotypic characterization of SWIB-family domain proteins

1.1. Gene expression analysis of T-DNA insertion mutants

T-DNA insertion lines were used as loss-of-function lines for the genes of interest. The effect

of the T-DNA insertions on the expression levels of the targeted genes needs to be evaluated

before we can characterize the T-DNA insertion lines.

Table 1: Overview of the T-DNA insertion lines used in the expression analysis and the leaf size analysis. The

genes that were targeted by the T-DNA insertion are shown together with the type of T-DNA insertion (GABI,

SAIL or SALK) that was used and the code that was given to the different lines.

In total 20 T-DNA insertion lines for the 7 genes of interest (SWIB1, SWIB2, SWIB3, SWIB4,

SWIB6, INT80 and INT90) were analyzed (Table 1). The plants were grown for 10 days and the

expression of SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 and INT90 in the different T-DNA

insertion lines was measured using quantitative real-time PCR or qRT-PCR.

At-code Gene T-DNA insertion line Code

At3g48600 SWIB1 SALK_008142 swib1-13, swib1-15

SALK_059703 swib1-24

SAIL_0903_E11 swib1-33


At3g48600 SWIB2 SAIL_0044_A03 swib2-1

SAIL_0913_G09 swib2-21

At4g34300 SWIB3 GABI_278H03 swib3-11

GABI_741D05 swib3-231, swib3-281

At4g35605 SWIB4 SAIL_1252_E08 swib4-1


SAIL_1156_C12 swib4-32


At2g35605 SWIB6 SALK_000628 swib6-14


At5g15980 INT80 GABI_803H10 int80-221, int80-292

SALK_008082 int80-33

At1g55890 INT90 SALK_200611 int90-1

Part 3: Results

21

First, we compared the expression levels of the 7 genes of interest in the wild type (figure 5-

H). The INT80, SWIB4 and SWIB6 genes showed a lower expression level compared to SWIB1,

SWIB2, SWIB3 and INT90.

There were five T-DNA insertion lines for the SWIB1 gene (figure 5-A). We observed a

complete knockout in the SWIB1 gene expression in two lines (> 99% downregulation, swib1-

13, and swib1-15). The SWIB1 expression was significantly downregulated (29%) in the swib1-

24 line. In the swib1-33 line, the SWIB1 gene expression was significantly upregulated (14%).

We used two T-DNA insertion lines for the SWIB2 gene and we observed a significant

downregulation in the SWIB2 gene expression in both of them (Figure 5-B). In the swib2-21

for example, we observed a downregulation of 68% in the SWIB2 gene expression.

There were three T-DNA insertion lines for the SWIB3 gene and all of them were significantly

downregulated in the SWIB3 gene expression (figure 5-C). In the swib3-281 line for example,

we observed a downregulation of 43% in the SWIB3 gene expression.

We used four T-DNA insertion lines for the SWIB4 gene (figure 5-D). We observed a significant

downregulation of 42% in the SWIB4 gene expression in the swib4-41 line. The swib4-2 and

the swib4-32 lines were significantly upregulated for the SWIB4 gene expression. In the swib4-

2 line for example, we observed an upregulation of 25% in the SWIB4 gene expression. The

swib4-1 line did not show any differences in the SWIB4 gene expression compared to the wild

type.

There were two T-DNA insertion lines for the SWIB6 gene (figure 5-E). The swib6-14 line was

significantly downregulated (29%) for the SWIB6 gene expression and the swib6-22 line was

significantly upregulated (13%) for the SWIB6 gene expression.

There were three T-DNA insertion lines for the INT80 gene and we observed a downregulation

in the INT80 gene expression in all of them (figure 5-F). In the int80-292 line for example, we

observed a significant downregulation (56%) in the INT80 gene expression.

There was one T-DNA insertion line for the INT90 gene and we observed a downregulation of

5% in the INT90 gene expression but this was not significant.

Part 3: Results

22

Figure 5: Relative expression levels of SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 and INT90 in their T-DNA

insertion lines. Each graph shows the different T-DNA insertion lines for which the expression levels were

measured and compared to the expression level in the wild type. The experiment was done in three biological

repeats. The error bars shown on the graphs represent the standard error.

Part 3: Results

23

1.2. Leaf size analysis of T-DNA insertion mutants

SWIB5 gain-of-function and loss-of-function lines produced smaller leaves caused by a

decrease in cell number, which was only partly compensated by an increase in cell size. SWIB5

belongs to the SWIB-family domain proteins and therefore it would be interesting to

characterize other members of this family and interacting partners of SWIB5 as well.

Therefore, T-DNA insertion lines were characterized to find out if SWIB1, SWIB2, SWIB3,

SWIB4, SWIB6, INT80 and INT90 have an influence on plant growth.

The plants were grown for 21 days, the leaves were harvested and ordered according to their

emergence in function of the time (cotyledons first, leaf one, leaf two, leaf three, etc.). The

size of each leaf was analyzed which allowed us to make a thorough comparison between the

T-DNA insertion mutants and the wild type. The third leaf that emerges from the SAM is the

first vegetative leaf for which growth is not dependent on seed reserves. Therefore we mostly

focused on the third leaf to compare differences in plant size.

The three lines for the T-DNA insertion of INT80 showed a similar pattern (Figure 6). There

was an overall decrease in leaf area in the different lines (int80-221, int80-292 and int80-33).

We observed a decrease in leaf area that was the strongest for the int80-221 line (14.1%, p

<0.0001) for the third leaf. The other two lines (int80-292 and int80-33) also showed a

significant decrease in leaf area (10.2%, p < 0.0001 and 8.4%, p < 0.01) of the third leaf. The

younger leaves appeared to be more affected although this was not always significant due to

a bigger variation in their size.

For the T-DNA insertion line of INT90 (int90-1) there was an overall increase in leaf area (figure

6). We observed an increase in leaf area of 7.5%, p < 0.01 of the third leaf. The older leaves

appeared to be more affected although this was not always significant.

The five lines for the T-DNA insertion of SWIB1 showed different patterns (figure 6). The swib1-

13, swib1-15 and swib1-47 lines showed no significant differences in leaf area of the third leaf.

The swib1-24 and swib1-33 lines did show a significant difference in leaf area of the third leaf.

The swib1-24 line showed a decrease in leaf area (13.2%, p < 0.0001) and the swib1-33 showed

an increase in leaf area (13.2%, p < 0.0001).

Part 3: Results

24

For the T-DNA insertion lines of SWIB2 (swib2-1 and swib2-21) there was no significant

difference in leaf area of the third leaf (figure 6). The younger leaves appeared to be

significantly larger for the swib2-1 line and significantly smaller for the swib2-21 line.

In the T-DNA insertion lines of SWIB3 there was a very similar pattern for two of the three

lines (swib3-231 and swib3-281, figure 6). In these two lines we observed a significant

decrease in leaf area of the third leaf. There was a decrease of 56.8% (p < 0.0001) for the

swib3-231 line and a decrease of 55.1% (p < 0.0001) for the swib3-281 line. The other leaves

appeared to be smaller as well. The 21 day old seedlings of the swib3-281 line were so small

that the eighth and the ninth leaf for this line had not come out yet at the time the leaves

were harvested. The swib3-11 line showed a significant increase in leaf area of the third leaf

(6.9%, p < 0.05) but there were no significant differences in leaf area of the other leaves.

In the T-DNA insertion lines of SWIB4 there was an overall increase in leaf area for three of

the four lines that were used (swib4-1, swib4-2 and swib4-32, figure 6). In these lines there

was a significant increase in leaf area of the third leaf. The swib4-1 line showed an increase of

10.3% (p < 0.01) and the swib4-2 line showed a very significant increase of 25.4% (p < 0.0001)

and the swib4-32 line showed an increase of 13.1% (p < 0.05) of the third leaf. The swib4-41

line showed no significant difference in leaf area of the third leaf but showed an overall

decrease in leaf area.

One of the SWIB6 T-DNA insertion lines (swib6-14) showed a significant increase in leaf area

of the third leaf (17.5% p < 0.0001, figure 6). The other leaves appeared to be larger as well

for the swib6-14 line. For the swib6-22 line we observed no significant differences in leaf area

for the third leaf.

Part 3: Results

25

Figure 6: Differences in leaf size for all leaves in the different T-DNA insertion mutants compared to the wild

type. The differences are displayed in percentage. A positive/negative value indicates that the T-DNA insertion

line has an increased/decreased leaf size compared to the wild type. For the cotyledons (cot) and leaf 1 and 2

(L1-2) the average leaf areas were taken as these leaves emerge simultaneously during plant development. The

table is color-coded: shades of green/red were used to display an increase/decrease in leaf area of the T-DNA

insertion line compared to the wild type. The p-values are displayed in the table as p < 0.05 (*), p < 0.01 (**) and

p < 0.0001 (***).

1.3. Detailed characterization of selected T-DNA insertion mutants

Four T-DNA insertion lines (swib3-281, swib4-2, swib6-14 and int80-221) were selected based

on the qRT-PCR results (figure 5) and the leaf analysis results (figure 7) for a more detailed

analysis. This more detailed analysis consists of a cellular analysis, a root analysis and a ploidy

analysis. These lines were also grown on different stress-inducing media followed by a leaf

size analysis.

Part 3: Results

26

Figure 7: The difference in leaf size between four selected mutants and the wild type. The average was taken

for the cotyledons (cot) and the first two leaves (L1-2). The error bars shown on the graphs represent the

standard error.

1.3.1. Cellular analysis

The cellular analysis was done to determine what the differences in the size of the third leaf

are caused by: a difference in the cell number, a difference in the epidermal cell size or a

combination of both. The plants were grown for 21 days in the growth chamber, the third leaf

was harvested and cell drawings of the abaxial epidermis were made to determine the leaf

cell number and cell size.

The third leaf of the swib3-281 line was significantly smaller (55%) compared to the wild type

line (figure 8-A). There were 43% fewer cells in the swib3-281 line and the pavement cells

were 27% smaller (figure 8-C, E). In conclusion, the decrease in leaf size of the swib3-281 line

was caused mostly by a decrease in the cell number but also by a decrease in the size of the

pavement cells.

Part 3: Results

27

In the swib4-2 line we observed a significant increase (25%) in the size of the third leaf (figure

8-A). There were 18% more cells in the swib4-2 line but there was no significant difference in

the average pavement cell size (figure 8-C, E). In conclusion, the increase in leaf size of the

swib4-2 line was caused by an increase in the cell number.

The third leaf of the swib6-14 line was significantly larger (18%) compared to the wild type

line (figure 8-A). There were 26% more cells in the swib6-14 line and the pavement cells were

18% smaller (figure 8-C, E). In conclusion, the increase in leaf size of the swib6-14 line was

caused by an increase in the cell number.

In the int80-221 line we observed a significant decrease (14%) in the size of the third leaf

(figure 8-B). There were 11% fewer cells in the int80-221 line but there was no significant

difference in the size of the pavement cells (figure 8-D, F). In conclusion, the decrease in leaf

size of the int80-221 line was caused by a decrease in the cell number.

Part 3: Results

28

Figure 8: Leaf size, leaf cell number and average pavement cell size of the third leaf of the T-DNA insertion

mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared to the wild type. The plants were grown for

21 days, the third leaf was harvested and cell drawings of the abaxial epidermis were made. The error bars shown

on the graphs represent the standard error.

Part 3: Results

29

1.3.2. Root analysis

The root analysis was done to find out if there is a similar phenotype in the root and the shoot.

The plants were grown for 12 days and the primary root length, the lateral root length and the

amount of lateral roots was evaluated.

Root development in the swib3-281 line was more significantly affected compared to the

other T-DNA insertion lines. There was a significant decrease in lateral root length (62%) and

in primary root length (40%) compared to the wild-type. Also the lateral root density was

significantly smaller (49%) compared to the wild type (figure 9-A, C, E).

For the T-DNA insertion line of SWIB4, we did not observe any differences in lateral root

length, primary root length and lateral root density compared to the wild type (figure 9-A, C,

E).

In the swib6 mutant, we found no significant difference in the lateral root length compared to

the wild type. We did however observe a significant increase in primary root length (24%) and

lateral root density (23%) compared to the wild type (figure 9-A, C, E).

For the T-DNA insertion line of INT80, we found no significant differences in lateral root length

and lateral root density compared to the wild type. We did however observe a small but

significant decrease in primary root length (10%) for this line (figure 9-B, D, F).

Part 3: Results

30

Figure 9: Lateral root length (LRL), primary root length (PRL) and lateral root density (LRD) of the T-DNA

insertion mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared to the wild type. Plants were grown

for 12 days and the LRL, the PRL and the LRD was evaluated. The error bars shown on the graphs represent the

standard error.

1.3.3. Ploidy analysis

The ploidy analysis was done to determine if differences in leaf size in the swib3, swib4, swib6

and int80 mutants could be linked to a difference in the ploidy level. During the

endoreduplication cycle, the genomic DNA is replicated without mitosis, resulting in an

increase in the ploidy level (2C, 4C, 8C and 16C nuclei). The plants were grown for 21 days in

Part 3: Results

31

the growth chamber and the third leaf was harvested and analyzed using the flow cytometer.

In the wild type, most of the nuclei were either in the 2C, 4C and 8C. We observed only a small

percentage of the wild type nuclei in the 16C (figure 10-A, B).

For the int80-221 line we observed an increase in the percentage of 4C nuclei and a similar

decrease in the percentage of 8C nuclei (figure 10-A) but this was not significant. In the swib3-

281 line there was an increase in the percentage of the 16C nuclei and a decrease in the

percentage of the 4C nuclei (figure 10-B) but again, this was not significant. For the swib4-2

line we observed an increase in the percentage of the 8C nuclei and a decrease in the

percentage of the 2C and the 4C nuclei (figure 10-B) but this was not significant as well. In the

swib6-14 line we observed no differences in the percentages of 2C, 4C, 8C and 16C nuclei

compared to the wild type (figure 10-B).

In conclusion, there were no significant differences in the percentages of 2C, 4C, 8C and 16C

nuclei in the mutant lines compared to the wild type.

Part 3: Results

32

Figure 10: Ploidy level of the T-DNA insertion mutants (swib3-281, swib4-2, swib6-14 and int80-221) compared

to the wild type. The third leaf was harvested for each T-DNA insertion mutant and the wild type and the ploidy

level was determined using the flow cytometer. The percentage of 2C, 4C, 8C and 16C is shown for each line. The

error bars shown on the graphs represent the standard deviation.

Part 3: Results

33

1.4. Phenotypic analysis of overexpression lines

Different independent T2 overexpression lines of SWIB1 and SWIB6 were grown for 28 days

in the growth chamber in soil to find out if there are differences in plant development

compared to the wild type. As these lines are in the T2 we expect ¼ to be homozygous, ½ to

be heterozygous for the p35S::GENE construct and ¼ to be wild type.

In the p35S::SWIB1 lines we observed plants that were smaller compared to the wild type

(figure 11). The leaves were curled in the abaxial direction and were not as elongated as the

wild type leaves.

Figure 11: Overexpression lines for the SWIB1 gene. The T2 segregating p35S::SWIB1 lines were grown in soil

for 28 days.

In the p35S::SWIB6 lines we also found plants that were smaller compared to the wild type

(figure 12) but the phenotype was more pronounced compared to the p35S::SWIB1 lines. The

leaves were also curled in the abaxial direction and were not as elongated as the wild type

leaves.

Part 3: Results

34

Figure 12: Overexpression lines for the SWIB6 gene. The T2 segregating p35S::SWIB6 lines were grown in soil for

28 days.

2. SWIB-domain family proteins under stress conditions

2.1. Gene expression analysis of a wild type line on stress

The expression levels of the genes of interest in a wild type line grown on stress-inducing

media were evaluated to determine their behavior under stress conditions.

Wild type plants were grown on ½ MS medium and after 10 days they were transferred to

different media containing stress-inducing compounds or to control medium. These different

media were used to subject the plants to osmotic stress, salt stress and oxidative stress. For

most of the stress-inducing media, two concentrations of the stress inducing compound were

used to mimic both mild and strong conditions. Mannitol (C6H14O6) was used to subject the

plants to osmotic stress. Salt (NaCl) was used to subject the plants to salt stress. Hydrogen

peroxide (H2O2) is a reactive oxygen species (ROS) and was used to subject the plants to

oxidative stress. We also used three compounds to subject the plants to oxidative stress in the

Part 3: Results

35

mitochondria or the chloroplasts. Rotenone (C23H22O6) is used as a broad-spectrum

insecticide, pesticide and piscicide since it inhibits the transfer of electrons from iron-sulfur

centers in complex I to ubiquinone (stress in the mitochondria). Paraquat (C12H14Cl2N2) is used

as a broad-spectrum herbicide. It uses the electrons produced by photosystem I in

chloroplasts to produce ROS (stress in the chloroplasts). Antimycin A (C28H40N2O9) is used as a

broad-spectrum piscicide. It inhibits the oxidation of ubiquinol in the electron transport chain

of oxidative phosphorylation (stress in the mitochondria).

On the medium containing 50 µM Antimycin A we observed a significant downregulation in

the SWIB2 and SWIB3 gene expression (figure 13-A). There was a significant upregulation in

the INT80 and INT90 gene expression.

On the medium containing 1 mM hydrogen peroxide (figure 13-B) we observed a significant

downregulation in the INT90, SWIB2, SWIB4, SWIB5 and SWIB6 gene expression. There was a

significant upregulation in the INT80 gene expression. On the medium containing 2 mM

hydrogen peroxide there was no significant downregulation for the genes of interest. We

found a significant upregulation in the INT80, SWIB1 and SWIB3 gene expression. We observed

a different response of the genes to both mild (1 mM) and strong (2 mM) conditions. The

genes showed a higher expression level on the strong concentration of H2O2 compared to the

expression level on the mild concentration.

On the medium containing 25 mM mannitol (figure 13-C) there was a significant

downregulation in the INT90, SWIB2, SWIB3, SWIB4, SWIB5 and SWIB6 gene expression. We

observed a significant upregulation in the INT80 and SWIB1 gene expression. On the medium

containing 100 mM mannitol we observed a significant downregulation in the INT90, SWIB2,

SWIB3, SWIB5 and SWIB6 gene expression. There was a significant upregulation in the SWIB1

gene expression.

On the medium containing 50 nM Paraquat (figure 13-D) we observed a significant

downregulation in the SWIB2, SWIB3, SWIB5 and SWIB6 gene expression. There was no

significant upregulation in the expression of the genes. On the medium containing 100 nM

Paraquat we observed a significant downregulation in the expression of all genes. There was

a similar response of the genes to both mild (50 nM) and strong (100 nM) conditions.

Part 3: Results

36

On the medium containing 10 µM rotenone (figure 13-E) we observed a significant

downregulation in the SWIB1, SWIB2, SWIB3, SWIB4, SWIB5, SWIB6 and INT80 gene

expression. There was a significant upregulation in INT90 gene expression on 10 µM rotenone.

On the medium containing 20 µM rotenone there was a significant downregulation in the

INT80 gene. We observed a significant upregulation for the SWIB1 and INT90 gene expression.

We observed a different response of the genes to both mild (10 µM) and strong (20 µM)

conditions. The genes showed a higher expression level on the strong concentration of

rotenone compared to the expression level on the mild concentration of rotenone.

On the medium containing 50 mM sodium chloride (figure 13-F) there was a significant

downregulation in the INT90, SWIB1, SWIB2, SWIB3, SWIB4, SWIB5 and SWIB6 gene

expression. We observed a significant upregulation in the INT80 gene expression. There was

a similar response of the genes to both mild (50 mM) and strong (150 mM) conditions.

In conclusion, we observed that the SWIB1, SWIB2, SWIB3, SWIB4, SWIB5, SWIB6, INT80 and

INT90 genes were all highly responsive to the different stress-inducing media.

Part 3: Results

37

Figure 13: Relative expression of the SWIB1, SWIB2, SWIB3, SWIB4, SWIB5, SWIB6, INT80 and INT90 genes on

different stress-inducing media. Seedlings of a wild type line were transferred from control medium to stress-

inducing medium after 10 days of growth. Each graph shows the relative expression levels of the genes of interest

on different stress-inducing media (10-20 µM rotenone, 50 µM Antimycin A, 1-2 mM hydrogen peroxide, 25-100

mM mannitol, 50-100 nM Paraquat and 50-150 mM sodium chloride). The error bars shown on the graphs

represent the standard error.

2.2. Leaf size analysis of selected T-DNA insertion mutants

It was already shown that defects in mitochondria or chloroplasts affect leaf development.

We want to determine if the swib3-281, swib4-2, swib6-14 and int80-221 mutants are more

tolerant or sensitive to stress. The plants were grown for 21 days on stress-inducing media,

Part 3: Results

38

the leaves were harvested and ordered according to their emergence in function of the time

(cotyledons first, leaf one, leaf two, leaf three, etc.). The size of each leaf was analyzed which

allowed us to make a thorough comparison between the T-DNA insertion lines and the wild

type. We mostly focused on the third leaf to compare differences in plant growth here as well.

First, the leaf size on stress-inducing media was compared to the leaf size on control medium

by calculating the ratio (figure 14).

Figure 14: The ratio of the leaf size on stress-inducing media to the leaf size on control medium.

Then these differences in leaf size in the T-DNA insertion mutants were compared to the wild

type by calculating the ratio (figure 15). This shows the increase or decrease in tolerance of

the T-DNA insertion mutants to the stress-inducing medium compared to the wild type.

Line Compound cot L1-2 L3 L4 L5 L6 L7 L8 L9

swib3-281 Antimycin A 0,78 0,62 0,34 0,29 0,19 0,15

Rotenone 0,82 0,54 0,45 0,38 0,24 0,21

Sodium chloride 0,78 0,29 0,24 0,20 0,19 0,16 0,38

Mannitol 0,58 0,25 0,29 0,28 0,42 0,53 0,81 0,93

Hydrogen peroxide 0,83 0,68 0,58 0,60 0,69 0,75 0,96 0,77 0,77

swib4-2 Antimycin A 0,75 0,77 0,57 0,61 0,46 0,35 0,26 0,16

Rotenone 0,70 0,43 0,20 0,14 0,04

Sodium chloride 0,69 0,31 0,23 0,23 0,31 0,35 0,31 0,30 0,28

Mannitol 0,45 0,35 0,30 0,28 0,36 0,51 0,52 0,49 0,85


swib6-14 Antimycin A 0,83 0,72 0,68 0,60 0,50 0,50 0,45 0,32

Rotenone 0,66 0,39 0,21 0,15 0,06 0,04

Sodium chloride 0,67 0,26 0,22 0,22 0,28 0,30 0,27 0,20 0,00

Mannitol 0,45 0,31 0,24 0,26 0,33 0,45 0,54 0,55 0,51


WT2 Antimycin A 0,85 0,85 0,65 0,53 0,53 0,47 0,53 0,44

Rotenone 0,70 0,39 0,27 0,19 0,10 0,05

Sodium chloride 0,74 0,35 0,24 0,24 0,25 0,31 0,21 0,14 0,33

Mannitol 0,58 0,29 0,23 0,25 0,30 0,36 0,55 0,38 0,20


int80-221 Antimycin A 0,83 0,92 0,66 0,63 0,52 0,41 0,33 0,65

Rotenone 0,84 0,48 0,32 0,24 0,12

Sodium chloride 0,82 0,37 0,31 0,29 0,33 0,32 0,19 0,22

Mannitol 0,76 0,30 0,28 0,28 0,36 0,41 0,51 0,35 0,24


WT1 Antimycin A 0,83 0,76 0,71 0,78 0,65 0,50 0,30 0,62

Rotenone 0,68 0,36 0,19 0,11 0,05

Sodium chloride 0,66 0,34 0,27 0,27 0,33 0,31 0,30 0,22 0,20

Mannitol 0,45 0,36 0,28 0,29 0,33 0,45 0,56 0,51 0,52


Part 3: Results

39

Figure 15: Differences in leaf size for all leaves in the selected T-DNA insertion lines for SWIB3, SWIB4, SWIB6

and INT80, compared to the wild type on different stress-inducing media containing 25 mM mannitol, 50 mM

NaCl, 10 µM rotenone, 1 mM H2O2 or 50 µM antimycin A. The differences are displayed in percentage. A

positive/negative value indicates that the T-DNA insertion line has an increased/decreased tolerance to the

medium compared to the wild type. For the cotyledons (cot) and leaf 1 and 2 (L1-2) the average leaf areas were

taken as these leaves emerge simultaneously in plant development. The table is color-coded: shades of green/red

were used to display an increase/decrease in leaf area of the T-DNA insertion line compared to the wild type.

The experiment was done in one biological repeat.

The swib3-281 line showed an increased sensitivity to Antimycin A (50 µM), an increased

tolerance to rotenone (10 µM), an increased sensitivity to salt stress (50mM NaCl), an

increased tolerance to Mannitol (25mM) and an increased sensitivity to hydrogen peroxide (1

mM). In conclusion, the swib3-281 line is highly responsive to stress-inducing media.

The swib4-2 line showed an increased sensitivity to Antimycin A (50 µM), an increased

sensitivity to rotenone (10 µM), no significant difference in sensitivity to salt stress (50mM

NaCl), an increased tolerance to Mannitol (25mM) and an increased tolerance to hydrogen

peroxide (1 mM). In conclusion, the swib4-2 mutant is also highly responsive to stress-inducing

media.

The swib6-14 line showed no significant difference in sensitivity to Antimycin A (50 µM), an

increased sensitivity to rotenone (10 µM), an increased sensitivity to salt stress (50mM NaCl),

no significant difference in sensitivity to Mannitol (25mM) and an increased tolerance to

Line Compound cot L1-2 L3 L4 L5 L6 L7 L8 L9

swib3-281 Antimycin A -9 -27 -47 -46 -65 -68

Rotenone 16 37 66 105 147 354

Sodium chloride 5 -17 1 -16 -25 -47 83

Mannitol 30 -30 3 -3 27 17 44 84

Hydrogen peroxide -16 -11 -15 -16 -19 -18 -7 6 107

swib4-2 Antimycin A -12 -10 -12 16 -12 -25 -51 -64

Rotenone -1 9 -27 -28 -60

Sodium chloride -7 -9 -4 -4 24 14 51 113 -15

Mannitol -23 23 30 13 22 41 -6 29 328

Mannitol -12 1 34 18 22 51 43 127 91

swib6-14 Antimycin A -3 -15 5 15 -5 7 -15 -28

Rotenone -6 0 -21 -22 -37 -14

Sodium chloride -10 -24 -7 -7 14 -4 32 40 0

Mannitol -23 8 3 5 11 22 -1 46 156

Hydrogen peroxide -17 -7 0 7 8 24 4 38 -1

int80-221 Antimycin A 0 21 -7 -19 -19 -17 10 5

Rotenone 23 -77 -97 -99 -100

Sodium chloride 23 11 15 9 0 3 -37 0

Mannitol 31 6 19 15 21 13 -8 -8 22

Hydrogen peroxide 31 12 -7 11 21 -8 36 33 15

Part 3: Results

40

hydrogen peroxide (1 mM). In conclusion, the swib6-14 line is highly responsive to Antimycin

A and rotenone, less so to the other stress-inducing media.

The int80-221 line showed an increased sensitivity to Antimycin A (50 µM), an increased

sensitivity to rotenone (10 µM), no significant difference in sensitivity to salt stress (50mM

NaCl), an increased tolerance to Mannitol (25mM) and an increased tolerance to hydrogen

peroxide (1 mM). In conclusion, the int80-221 mutant is also highly responsive to Antimycin A

and rotenone, less so to the other stress-inducing compounds.

3. Expression analysis of SWIB5

The expression analysis of SWIB5 was done to determine where the gene is expressed during

the early development of Arabidopsis thaliana using independent pSWIB5::GFP::GUS lines.

The seedlings were harvested on day 5, 9 and 12. The first time point (day 5) was chosen to

follow the early development of the first two leaves. The other time points (Day 9 and Day 12)

were chosen to further follow the development of the first two leaves and the other tissues.

The seedlings were imaged using a DMLB binocular (Leica) fitted with a camera.

Overall, the expression of the pSWIB5::GFP::GUS construct was low. On day 5 there was GUS

expression in the first two leaves (figure 16-A) and most of it was at the edges of the leaves.

We observed GUS expression in the primary root and the root hairs starting from the

hypocotyl (figure 16-D). We also found GUS expression in the root tip (figure 16-G).

On day 9 there was also GUS expression in the first two leaves (figure 16-B) and most of it was

at the edges of the leaves as well. We observed GUS expression in the primary root and the

root hairs starting from the hypocotyl (figure 16-E). We also found GUS expression in the root

tip (figure 16-H). The GUS expression was the highest in the proliferating cells near the

quiescent center (QC) but there was also GUS expression in the expanding cells.

On day 12 there was no more GUS expression in the first two leaves (figure 16-C) or in the

primary root and the root hairs starting from the hypocotyl of the primary root (figure 16-F).

We did observe GUS expression in the root tip (figure 16-I).

Part 3: Results

41

Figure 16: pSWIB5::GFP::GUS expression in the 5, 9 and 12 day old seedlings. The first two leaves (A, B, C), the

start of the primary root (D, E, F) and the root tip (G, H, I) are shown.

4. Localization analysis of SWIB-family domain proteins

This experiment was done to confirm the localization of the SWIB-family domain proteins and

to verify that the p35S::GENE::GSGreen constructs are functional. SWIB2, SWIB3 and SWIB4

are targeted to the chloroplasts where they associate with the nucleoid, SWIB4 is also targeted

to the nucleus, SWIB5 is targeted to the mitochondria and SWIB6 is targeted to both the

chloroplasts and the mitochondria. SWIB1 is targeted to the cytoplasm (Melonek et al, 2012).

Part 3: Results

42

Using the SUBcellular localization database for Arabidopsis (SUBA3), we looked up the

predicted location of the interacting partners of SWIB5. INT80 was predicted to be targeted

to the cytosol, the mitochondria, the plastids and the nucleus. INT90 was predicted to be

targeted to the cytosol, the mitochondria and the plastids.

We transiently expressed different p35S::GENE::GSGreen constructs for the SWIB1, SWIB2,

SWIB3, SWIB4, SWIB6, INT80 and INT90 genes in Nicotiana benthamiana leaves. Different

markers for mitochondria and chloroplasts and a P19 anti-viral RNA silencing were co-

infiltrated with the different constructs. When the plants were four weeks old the leaves were

infiltrated using Agrobacterium tumefaciens and imaged four to six days later. A piece of the

leaf that was infiltrated was analyzed using a confocal scanning laser microscope (CSLM).

With the construct for SWIB1 the expression of GFP was low (figure 17-A, B). We did not

observe co-localization with the mitochondrion marker (figure 17-A) and the chloroplast

marker (figure 17-B). SWIB1 is targeted to the cytoplasm so we did not expect co-localization.

Using the p35S::SWIB2::GSGreen construct we observed co-localization with the chloroplast

marker (Figure 17-C). SWIB2 is targeted to the chloroplasts so we expected co-localization.

With the construct for SWIB3 we observed co-localization with the chloroplast marker (Figure

17-D). SWIB3 is targeted to the chloroplasts so we expected co-localization.

Using the p35S::SWIB4::GSGreen construct we observed co-localization with the chloroplast

marker (Figure 17-E). SWIB4 is targeted to the chloroplasts and the nucleus so we expected

co-localization with the chloroplast marker. However, there was no expression in the nucleus.

With the p35S::SWIB6::GSGreen construct we did not observe co-localization with the

mitochondrion marker (figure 17-F) or the chloroplast marker (Figure 17-G). SWIB6 is targeted

to the mitochondria and the chloroplasts so we expected co-localization with both markers.

Using the p35S::INT80::GSGreen there was no co-localization with the mitochondrion marker

(figure 17-H). We did observe co-localization with the chloroplast marker (Figure 17-I).

With the p35S::INT90::GSGreen construct there was no co-localization with the mitochondrion

marker (Figure 17-J) as there was no GFP expression. We did observe co-localization with the

chloroplast marker (Figure 17-K).

Part 3: Results

43

Figure 17: CLSM image of Nicotiana benthamiana leaves after co-infiltration of the p35S::GENE::GSGreen

construct with the mitochondrion or the chloroplast marker. p35S::SWIB1::GSGreen with the mitochondrion

marker (A) and the chloroplast marker (B). p35S::SWIB2::GSGreen with the chloroplast marker (C).

p35S::SWIB3::GSGreen with the chloroplast marker (D). p35S::SWIB4::GSGreen with the chloroplast marker (E).

p35S::SWIB6::GSGreen with the mitochondrion marker (F) and the chloroplast marker (G). p35S::INT80::GSGreen

with the mitochondrion marker (H) and the chloroplast marker (I). p35S::INT90::GSGreen with the mitochondrion

marker (J) and the chloroplast marker (K). A magnification of 60 times was used.

Part 3: Results

44

5. Interaction analysis of SWIB5

A tandem affinity purification (TAP) experiment was done to find new interacting partners of

SWIB5 which could give us a better understanding of the function of SWIB5 at the molecular

level. Two independent p35S::SWIB5::GSGreen lines were used (table 2). GSGreen is a new tag

that contains two different domains which can be cut off separately in two enzymatic or

chemical reactions and is therefore very useful in TAP. The TAP was done on 6 day old

seedlings of the p35S::SWIB5::GSGreen lines. Interacting partners were found in only one of

the two independent p35S::SWIB5::GSGreen lines.

Table 2: The proteins found in the tandem affinity purification experiment using p35S::SWIB5::GSGreen

seedlings.

AT1G20950 encodes a phosphofructokinase protein. Submitting the gene to SUB3A showed

that it is predicted to be located in the mitochondria, the nucleus, the cytosol and the

extracellular space. The gene expression of AT1G20950 is strongly responsive to sucrose

(Gonzali et al, 2006).

AT3G58140 is a phenylalanyl-tRNA synthetase. Submitting the gene to SUB3A showed that it

is predicted to be located in the mitochondria, the plastids, the cytosol, the plasma membrane

and the extracellular space. Using The Arabidopsis Information Resource database (TAIR), we

found it to be involved in chloroplast organization, isopentenyl diphosphate biosynthetic

process, methylerythritol 4-phosphate pathway, ovule development and phenylalanyl-tRNA

aminoacylation (TAIR).

AT1G55450 is an S-adenosyl-L-methionine-dependent methyltransferases superfamily

protein. Submitting the gene to SUB3A showed that it is predicted to be located in the

mitochondria, the cytosol and the nucleus. It is involved in methylation and response to salt

stress (TAIR).

At-code Protein description

At1g31760 SWIB/MDM2 domain superfamily protein

At1g20950 Phosphofructokinase family protein

At3g58140 phenylalanyl-tRNA synthetase class IIc family protein

At1g55450 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein

At3g24430 HCF101 | ATP binding

Part 3: Results

45

AT3G24430 is a high chlorophyll fluorescence 101 protein. Submitting the gene to SUB3A

showed that it is predicted to be located in the mitochondria, the plastids, the cytosol and the

nucleus. It is involved in carotenoid biosynthetic process, chlorophyll biosynthetic process,

chloroplast relocation, iron-sulfur cluster assembly, isopentenyl diphosphate biosynthetic

process, methylerythritol 4-phosphate pathway, maltose metabolic process, ncRNA metabolic

process, pentose-phosphate shunt, photosynthesis, positive regulation of catalytic activity,

rRNA processing, starch biosynthetic process, thylakoid membrane organization (TAIR).

Part 4: Discussion

46

Part 4: Discussion

1. Aim of the research project

In this research project we studied the six members of the SWIB-domain protein family

(SWIB1, SWIB2, SWIB3, SWIB4, SWIB5 and SWIB6) and two interacting partners of SWIB5

(INT80 and INT90). The characterization of these genes was divided into three work packages

(WP). In WP1 loss- and gain-of-function lines were characterized phenotypically. In WP2, we

evaluated the response of these genes to stress-inducing compounds. Also, loss-of-function

lines under stress conditions were characterized phenotypically. In WP3 the localization of the

proteins of interest was studied. We also looked for new interacting partners of SWIB5.

2. Localization analysis of SWIB-family domain proteins

It was previously shown that SWIB2, SWIB3 and SWIB4 are targeted to the chloroplasts, SWIB5

to the mitochondria and SWIB6 to both organelles. SWIB1 is targeted to the cytoplasm

(Melonek et al, 2012). The localization of SWIB2, SWIB3 and SWIB4 was confirmed in the co-

localization experiment using Nicotiana benthamiana. SWIB1 and SWIB6 were not confirmed

and this might be due to the low signal we observed for those p35S::GENE::GSGreen

constructs. The functionality of the p35S::GENE::GSGreen constructs were verified as well.

Using the SUBcellular localization database for Arabidopsis proteins (SUBA3), INT80 was

predicted to be targeted to the cytosol, the mitochondria, the plastids and the nucleus and

INT90 was predicted to be targeted to the cytosol, the mitochondria and the plastids. For both

INT80 and INT90, the localization in the chloroplasts was confirmed. As they are interacting

partners of SWIB5, they should be localized to the mitochondria as well but this was not

confirmed. It is possible that the co-transformation with Agrobacterium tumefaciens failed

and that the GFP signal was due to autofluorescence in the Nicotiana benthamiana leaves.

Part 4: Discussion

47

3. Phenotypic characterization of SWIB-family domain proteins

The phenotypic characterization was done using loss- and gain of function lines to find out if

the SWIB-family domain proteins play a role in leaf development.


The gene expression analysis of T-DNA insertion lines showed that for each gene of interest,

except for INT90, there was at least one T-DNA insertion line that showed significant

downregulation in the expression level of the corresponding gene. For some T-DNA insertion

lines, there was a significant upregulation in the corresponding gene. It is possible that a

truncated version of the gene is upregulated. In the swib4-2 line for example, there was 25%

upregulation in the expression level of the SWIB4 gene. The T-DNA insertion (SAIL_1298_E08)

was located in an exon so it is likely that the protein is truncated and therefore not functional.

In the leaf size analysis of the T-DNA insertion lines of SWIB1, SWIB2, SWIB3, SWIB4, SWIB6,

INT80 and INT90, we found mutants both with smaller and larger leaves. The swib3-281 line

showed the strongest decrease in leaf size and the swib4-2 line showed the strongest increase

in leaf size. There were different independent T-DNA insertion lines for each gene of interest

and these independent lines always showed a similar pattern of leaf size. Nevertheless, the

phenotype of some independent lines was more pronounced than that of others and this

could often be linked to the change in expression of the gene of interest. In the T-DNA

insertion lines for SWIB3 for example, we found a strong decrease in leaf size in the swib3-281

line but no noteworthy differences in the swib3-11 line. The expression level of SWIB3 was

much lower in the swib3-281 line compared to the swib3-11 line, which could explain the

difference in the phenotype. In the T-DNA insertion lines of SWIB4, there was only one line for

which SWIB4 was downregulated (swib4-41) and this was also the only line that showed a

decrease in leaf size compared to the wild type. Since we observed a clear phenotype in the

swib3-281, swib4-2, swib6-14 and int80-221 lines, they were characterized in detail.

Part 4: Discussion

48

3.2. Detailed characterization of selected T-DNA insertion mutants

We found an overall decrease in leaf size for the swib3-281 line and the cellular analysis

showed that there were much fewer cells in this line and that the pavement cells were also

smaller compared to the wild type. Therefore, it is possible that SWIB3 is involved in the

regulation of both cell proliferation and expansion (Gonzalez et al, 2012). In the root analysis

we found a significant decrease in lateral and primary root length compared to the wild type.

Also the lateral root density was significantly lower compared to the wild type so next to the

shoot phenotype there was also a pronounced root phenotype in the swib3 mutant. Since

SWIB3 is targeted to the chloroplasts and since we saw both a root and shoot phenotype, it is

possible that it plays an important role in energy production. A swib3 mutant would then have

a defect in energy production which would explain the decreased growth in the whole plant.

It was previously shown that in gain-of-function lines of SWIB4 the cotyledons are smaller

compared to the wild type (Melonek et al, 2012). We observed an overall increase in leaf size

for the swib4-2 line and the cellular analysis showed that this was due to an increased cell

number. This means that SWIB4 might play a role in cell proliferation. Since SWIB4 is targeted

to the chloroplasts and the nucleus and since we only found a shoot phenotype, it is possible

that it plays an important role in cell cycle regulation as the root would also be affected if

SWIB4 was important in energy production. Because a gain of function of SWIB4 results in

smaller leaves and a loss-of-function in larger leaves it is possible that SWIB4 is a negative

regulator of the cell cycle.

In gain-of-function lines of SWIB6 the leaves are smaller compared to the wild type. We found

an overall increase in leaf size for the swib6-14 line and the cellular analysis showed that this

was due to an increased cell number. Therefore, it is possible that SWIB6 plays a role in cell

proliferation. We observed a significant increase in the primary root length and the lateral

root density was also higher compared to the wild type. Since SWIB6 is targeted to the

mitochondria and the chloroplasts and since we saw both a root and shoot phenotype, it is

possible that it plays an important role in energy production. Because a gain of function of

SWIB6 results in smaller leaves and a loss-of-function in larger leaves, it is possible that SWIB6

is a negative regulator of energy production, which would explain the increased growth in the

whole plant.

Part 4: Discussion

49

We observed an overall decrease in leaf size for the int80-221 line and the cellular analysis

showed that this was due to a decreased cell number. Therefore, INT80 may play a role in cell

proliferation. We observed a significant decrease in primary root length compared to the wild

type so there was a root phenotype next to the shoot phenotype. Since INT80 is targeted to

both mitochondria and chloroplasts and since it interacts with SWIB5, it is possible that it is

involved in energy production. This would also explain the decreased growth in both the shoot

and the root.

All four lines show a difference in leaf size that is predominantly caused by a difference in cell

number. Therefore it is possible that SWIB-family domain proteins play an important role in

cell cycle regulation.

4. Gene expression analysis of SWIB-family domain proteins

In the third leaf of 10 day old wild type plants, the SWIB4, SWIB6 and INT80 genes showed

lower expression levels compared to the SWIB1, SWIB2, SWIB3 and INT90 genes. It is possible

that the expression of SWIB4, SWIB6 and INT80 is cell type or tissue specific and that SWIB1,

SWIB2, SWIB3 and INT90 are expressed in all cells. This would explain these differences as the

expression levels were measured in the whole third leaf. We could evaluate the expression

levels of the genes of interest in pGENE::GFP::GUS lines to determine if the expression is cell

or tissue specific. Another explanation could be that the higher expression levels of SWIB1,

SWIB2, SWIB3 and INT90 are due to their role in cell expansion as the expression analysis was

done in expanding leaves.

In the expression analysis of SWIB5 using a pSWIB5::GFP::GUS line we found that SWIB5 was

expressed at the edges of the first two leaves during early development (day 5 and 9). When

the leaves were expanding (day 12) there was no more expression of SWIB5. In the root tip,

we observed SWIB5 expression at all three time points (day 5, 9 and 12) and the highest

expression of SWIB5 was observed in the cells surrounding the quiescent center. This means

that SWIB5 is expressed in cells that are proliferating and that it might play a role in cell

proliferation. During the development of root hairs we observed expression of SWIB5 (day 5

and 9) and at day 12 there was no more expression in the root hairs. As the root hairs are only

composed out of one cell, it is possible that we observed SWIB5 expression when they were

Part 4: Discussion

50

actively expanding. This means that SWIB5 might also be involved in cell expansion (Tominaga-

Wada et al, 2011). Studying the development of root hairs in loss-of-function lines for all the

SWIB-family domain proteins could give us a better understanding of their role in cell

expansion. It is likely that SWIB5 is expressed in cells that are proliferating or that are actively

expanding as we observed most of the expression in the developing parts of the plant. Using

the Arabidopsis eFP Browser, it was also shown that SWIB5 is expressed in the developing

parts of the plant. As SWIB5 might be involved in organelle gene expression regulation, it is

possible that it is a regulator of plant growth.

5. SWIB-family domain proteins under stress conditions

The characterization of the SWIBs on stress conditions was done to find out if they are involved

in the stress response and if they are more tolerant or sensitive to different stress conditions

including stress in mitochondria and chloroplasts.

5.1. Gene expression analysis of a wild type line on stress

The genes of interest were highly responsive to osmotic, salt, and oxidative stress and there

was an overall downregulation in their expression levels except for the INT80 gene. The INT80

gene showed significant upregulation on the stress-inducing media containing Antimycin A,

hydrogen peroxide and sodium chloride. On the media containing hydrogen peroxide and

rotenone, but mostly on the medium containing rotenone, we observed a difference in

expression levels for the mild and strong stresses of the compound. On the medium containing

rotenone for example, there was an overall downregulation of the genes under mild

conditions and no differential expression (SWIB2, SWIB4, SWIB6 and INT80) or upregulation

(SWIB1, SWIB3, SWIB5 and INT90) under strong conditions. Under mild conditions the plants

are still able to grow and complete their lifecycle. Under strong conditions however, plants

are not able to grow and will eventually die. These two different regulatory states of the plant

might be the reason we observed differences in expression levels under mild and strong

conditions.

Part 4: Discussion

51

Antimycin A induces stress in the mitochondria by inhibiting the oxidation of ubiquinol in the

electron transport chain of oxidative phosphorylation (Sweetlove et al, 2002). It also induces

stress in the chloroplasts by inactivation of the chloroplast ndhB gene (Taira et al, 2013). Upon

treatment with Antimycin A, we observed downregulation for the genes that are targeted to

the chloroplasts (SWIB2, SWIB3 and SWIB4), upregulation for the gene that is targeted to the

mitochondria (SWIB5) and no differential expression for the gene that is targeted to both

(SWIB6). It was previously shown that some genes in the mitochondria are differentially

expressed upon Antimycin A treatment. It is possible that Antimycin A influences the

expression levels of the SWIBs targeted to the mitochondria, thereby changing the expression

levels of the genes in the mitochondria. It was also shown that dysfunctional chloroplasts

induce upregulation of the expression of the mitochondrion genes in Arabidopsis (Liao et al,

2015). As Antimycin A also induces stress in chloroplasts it is possible that thereby

upregulation is induced in the expression levels of mitochondrion genes.


The leaf size analysis of the selected mutant lines (swib3-281, swib4-2, swib6-14 and int80-

221) showed that they are often more sensitive or tolerant to stress-inducing media.

Rotenone induces stress in mitochondria by inhibiting the transfer of electrons from iron-

sulfur centers in complex I to ubiquinone. The swib4-2, swib6-14 and int80-221 lines are more

sensitive to rotenone than the wild type. SWIB6 and INT80 are targeted to the mitochondria

so it is not surprising that the mutant lines for these genes are more sensitive to stress in

mitochondria. As SWIB4 is targeted to the chloroplasts and the nucleus, it is surprising that

the mutant line for this gene is more sensitive to rotenone. However, it is possible that SWIB4

is indirectly involved in the stress response to rotenone. The swib3-281 line on the other hand

is more tolerant to rotenone than the wild type. As SWIB3 is targeted to the chloroplasts it is

not logic that the mutant line for this gene is more tolerant to stress in mitochondria.

However, it might be involved indirectly in the stress response to rotenone.

All of the selected mutant lines were more sensitive to Antimycin A treatment than the wild

type. As Antimycin A induces stress in both mitochondria and chloroplasts it is not surprising

Part 4: Discussion

52

that swib3, swib4, swib6 and int80 mutants are more sensitive to Antimycin A treatment

because they are all targeted to mitochondria and/or chloroplasts.

The swib3-281, swib4-2, swib6-14 and int80-221 lines were found to be more tolerant to

osmotic stress compared to the wild type. Therefore it is unlikely that SWIB3, SWIB4, SWIB6

and INT80 play a role in the response to osmotic stress.

6. Interaction analysis of SWIB5

The interaction analysis was done to discover new interacting partners of SWIB5 as this might

give us a better understanding of the function of SWIB5 at the molecular level.

Because the tandem affinity purification only identified proteins in one of the two lines that

were used, the experiment will be repeated in the future to confirm the results. Repeating the

experiment might also confirm the interacting partners of SWIB5 that were characterized in

this research (INT80 and INT90) as these were not identified in this purification. It is possible

that INT80 and INT90 were not confirmed because they might only transiently interact with

SWIB5 and were therefore missed in this experiment or because the previous TAP used a cell

culture and this TAP used in planta material. The TAP resulted in the identification of four

possible interacting partners of SWIB5. As two of the proteins are involved in chloroplast

organization or the biosynthesis of chlorophyll it is possible that SWIB5 is involved in the

biosynthesis of chloroplasts. This is not logic because SWIB5 is targeted to the mitochondria.

However, it is possible that SWIB5 is indirectly involved in the biosynthesis of chloroplasts.

One of the proteins that was discovered is a methyltransferase. It is possible that this protein

is recruited by SWIB5 to remodel the chromatin through DNA methylation. We are not certain

that the proteins that were discovered are true interacting partners of SWIB5. The interacting

partners should therefore be confirmed in another experiment.

Part 4: Discussion

53

7. Conclusion

In conclusion, we found that loss-of-function lines for different SWIBs result in changes in the

leaf size and that SWIB3, SWIB4, SWIB6 and INT80 play a role in cell cycle regulation. A swib3

loss-of-function mutant showed a strong decrease in leaf size whereas swib4 and swib6 loss-

of-function mutants produced larger leaves. A SWIB6 gain-of-function mutant produced

smaller leaves that were curled up. It was also shown that SWIB5 is expressed in cells that are

proliferating and actively dividing and the interaction analysis showed that SWIB5 might play

a role in chloroplast biosynthesis and/or chromatin remodeling by DNA methylation. We also

showed that the SWIBs are highly responsive to stress in mitochondria and chloroplasts at the

transcription level and that swib mutants are often more sensitive to these stresses which

results in smaller leaves.

Deel 4: Discussie

54

Deel 4: Discussie

De localisatie van SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 en INT90 werd onderzocht met

behulp van een p35S::GEN::GSGreen construct voor de verschillende genen. Dit construct

werd samen met merkers voor de mitochondriën en chloroplasten geïnfiltreerd in Nicotiana

benthamiana. De reeds aangetoonde localisatie van SWIB2, SWIB3 en SWIB4 (chloroplasten)

werd bevestigd in dit experiment. Voor SWIB1 (cytoplasma) en SWIB6 (mitochondriën en

chloroplasten) werd dit niet bevestigd. We toonden aan dat INT80 en INT90 naar de

chloroplasten worden gestuurd. Deze interactoren van SWIB5 zouden ook naar de

mitochondriën moeten gestuurd worden aangezien SWIB5 daar naartoe gaat, maar dit werd

niet bevestigd.

T-DNA insertie mutanten voor de genen van interesse werden fenotypisch gekarakteriseerd

om te bepalen of de SWIBs een rol spelen in bladontwikkeling. Door middel van een qRT-PCR

werd het expressieniveau van de genen in hun T-DNA insertie lijnen bepaald. De swib3-281

lijn bijvoorbeeld produceerde kleinere bladeren en de swib4-2 en swib6-14 lijnen

produceerden grotere bladen in vergelijking met het wild type.

In de swib3-281 lijn lag het aantal en de grootte van de cellen aan de basis van de kleinere

bladgrootte, dus het is mogelijk dat SWIB3 een rol speelt in celdeling en celexpansie.

Aangezien we ook een wortelfenotype waarnamen in deze lijn en SWIB3 naar de chloroplasten

wordt gestuurd, is het mogelijk dat SWIB3 een belangrijke rol speelt in energieproductie.

Ook SWIB4 speelt een rol in celdeling aangezien de grotere bladeren in de mutant te wijten

waren aan een groter aantal cellen. Het werd reeds aangetoond dat een gain-of-function lijn

voor SWIB4 kleinere bladeren produceert. Aangezien we in de loss-of-function lijn grotere

bladeren waarnamen, is het dus mogelijk dat SWIB4 een negatieve regulator is van de

celcyclus.

De SWIB6 gain-of-function lijn produceerde kleinere bladeren en de loss-of-function lijn

produceerde grotere bladeren. De oorzaak van dit verschil in bladgrootte met het wild type

was een verschil in het aantal cellen. De swib6 mutant vertoonde ook een wortelfenotype

naast het scheutfenotype, dus het is mogelijk dat SWIB6 een negatieve regulator is van

energieproductie.

Deel 4: Discussie

55

In de int80 mutant zagen we kleinere bladeren als gevolg van een kleiner aantal cellen dus is

het mogelijk dat ook INT80 betrokken is in celdeling. Omdat we naast het sheutfenotype ook

een wortelfenotype waarnamen, is het mogelijk dat INT80 een rol speelt in energie productie

in de mitochondriën, aangezien het daar interageert het met SWIB5.

In de expressie analyse van SWIB5 met behulp van pSWIB5::GFP::GUS lijnen toonden we aan

dat SWIB5 vooral tot expressie komt in cellen en weefsels die actief delen en expansie

ondergaan. Zo zagen we GUS expressie op dag 5 en 9 in de worteltip en op de rand van de

eerste twee bladeren. Op dag 12 waren de eerste twee bladeren niet meer actief aan het

delen en we zagen dan ook geen GUS expressie meer. In de worteltip was de GUS expressie

het hoogst in de buurt van het QC, dus is het niet onwaarschijnlijk dat SWIB5 een belangrijke

rol speelt in ontwikkelende delen van de plant. In de wortelharen werd er ook GUS expressie

waargenomen op dag 5 en 9 dus is het mogelijk dat SWIB5 ook een rol speelt in cel expansie

aangezien wortelharen slechts uit 1 cel bestaan die expansie ondergaat.

De expressie van de genen van interesse onder stress condities werd onderzocht om te

bepalen of ze een rol spelen in de stress respons. We toonden aan dat hun expressieniveau

sterk beïnvloed door stress condities. Met als enige uitzondering het INT80 gen, zagen we

voor alle genen van interesse neerregulatie als gevolg van groei op stress-inducerend medium.

Antimycin A is een compound die stress induceert in de mitochondriën en de chloroplasten.

Als planten werden gegroeid op medium dat Antimycin A bevat, zagen we neerregulatie van

de genen die naar de chloroplasten worden gestuurd (SWIB2, SWIB3 en SWIB4), opregulatie

van het gen dat naar de mitochondriën wordt gestuurd (SWIB5) en geen verschil in expressie

in het gen dat naar beide celorganellen wordt gestuurd (SWIB6). Het is daarom mogelijk dat

Antimycin A zorgt voor neerregulatie van de SWIBs die naar de chloroplasten gestuurd

worden. Het werd reeds aangetoond dat defecten in chloroplasten voor opregulatie van

genen in de mitochondriën kunnen zorgen. Het is dus mogelijk dat de genen in de

mitochondriën opgereguleerd worden als gevolg van neerregulatie van de SWIBs die naar de

chloroplasten gestuurd worden.

De bladgrootte van de T-DNA insertie mutanten werd ook gekarakteriseerd onder

stresscondities. Het is op deze manier mogelijk om te bepalen of bepaalde mutanten meer

tolerant of gevoelig zijn voor een compound. Rotenone zorgt voor stress in de mitochondriën

en de swib4-2, swib6-14 en int80-221 lijnen waren minder tolerant tegen deze compound. Dit

Deel 4: Discussie

56

is niet verrassend voor SWIB6 en INT80, aangezien deze naar de mitochondriën worden

gestuurd. Voor SWIB4 was dit wel onverwacht, aangezien het naar de chloroplasten en de

nucleus wordt gestuurd. Het is wel mogelijk dat SWIB4 een indirecte rol speelt in de

stressrespons op rotenone. De swib3-281 lijn was minder gevoelig tegen rotenone in

vergelijking met het wild type. Dit was onverwacht maar het is mogelijk dat ook SWIB3 een

indirecte rol speelt in de stress respons op rotenone. Op Antimycin A zagen we in alle

mutanten verhoogde gevoeligheid en dit is was verrassend aangezien SWIB3, SWIB4, SWIB6

en INT80 naar de mitochondriën en/of de chloroplasten worden gestuurd en Antimycin in

beide celorganellen stress induceert.

Voor de interactie analyse van SWIB5 werd een TAP experiment uitgevoerd met als doel

nieuwe interagerende partners van SWIB5 te vinden. De INT80 en INT90 interacties werden

niet bevestigd maar dit kan te wijten zijn aan het materiaal dat werd gebruikt. In deze TAP

werd namelijk gebruik gemaakt van in planta terwijl in de vorige TAP een celcultuur werd

gebruikt. De interagerende partners van SWIB5 die werden ontdekt, geven ons reden om te

geloven dat SWIB5 betrokken is in de biosynthese van chloroplast materiaal en in DNA

methylatie.

We hebben dus aangetoond dat SWIB3, SWIB4, SWIB6 en INT80 mogelijks een rol spelen in

regulatie van de cel cyclus en dat SWIB3, SWIB4 en SWIB6 een rol zouden kunnen spelen in

energieproductie. Er werd ook getoond dat SWIB5 vooral tot expressie komt in ontwikkelende

delen van de plant en dat het een rol zou kunnen spelen in biosynthese van chloroplast

materiaal en DNA methylatie. De expressie van de SWIB genen wordt sterk beïnvloed door

stresscondities en swib mutanten zijn over het algemeen gevoeliger aan stress.

Part 5: Material and methods

57


1. Material

1.1. Plant material

The seeds of different T-DNA insertion lines for SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80

and INT90 and wild type seeds were used. A genotypic analysis was done and only the plants

that were homozygous for the T-DNA insertion were used in the following analyses. Table 3

gives an overview of the gene that was targeted by the T-DNA insertion, the type of T-DNA

insertion (GABI, SAIL or SALK) that was used and the code that was given to the lines that were

analyzed. All of the Arabidopsis thaliana lines (col-0 background) were grown on the same

trays to ensure homogeneity in growth behavior as this is an important factor in phenotypic

analyses.

Table 3: Overview of the T-DNA insertion lines for the genes of interest. The genes that were targeted by the

T-DNA insertion are shown together with the type of T-DNA insertion (GABI, SAIL or SALK) that was used and the

code that was given to the different lines.

At-code Gene T-DNA insertion line Code

At3g48600 SWIB1 SALK_008142 swib1-13, swib1-15




At3g48600 SWIB2 SAIL_0044_A03 swib2-1

SAIL_0913_G09 swib2-21

At4g34300 SWIB3 GABI_278H03 swib3-11

GABI_741D05 swib3-231, swib3-281

At4g35605 SWIB4 SAIL_1252_E08 swib4-1


SAIL_1156_C12 swib4-32


At2g35605 SWIB6 SALK_000628 swib6-14


At5g15980 INT80 GABI_803H10 int80-221, int80-292

SALK_008082 int80-33

At1g55890 INT90 SALK_200611 int90-1


58

The seeds of different independent overexpression lines for SWIB3 and SWIB6 were used for

phenotypic characterization. The seeds that were used were derived from a T2 stock (second

transgenic generation, heterozygous) as the T3 (third transgenic generation, homozygous)

stock was still growing and could not be harvested yet. Table 4 gives an overview of the gene

that was overexpressed and the code that was given to the lines that were analyzed.

Table 4: Overview of the codes used for the lines containing the p35S::GENE constructs.

For the localization analysis different pSWIB5::GFP::GUS lines were used. Table 5 gives an

overview of the codes used for the different lines containing the pSWIB5::GFP::GUS construct.

Table 5: Overview of the codes used for the lines containing the pSWIB5::GFP::GUS construct.

1.2. Growth media

To grow the seeds, petri dishes were used (Millipore Corp.) containing 100 mL of ½ MS

medium for round petri dishes and 70 mL of ½ MS medium for square petri dishes. The

medium was made by adding 2.15 g/L MS Salt (Duchefa Biochemie), 0.5 g/L MES buffer

(Duchefa biochemie) and 10 g/L sucrose to purified water. The pH was set at 5.8 using 1 M

At-code Construct Code

At3g48600 p35S::SWIB1 10212.1

10212.2

10212.3

At2g35605 p35S::SWIB6 10213.1

10213.2

10213.3

At-code Construct Code

At1g34760 pSWIB5::GFP::GUS 1.1.1

1.1.2

2.2.1

2.2.2

4.3.2

4.3.3


59

NaOH and 9 g/L Plant Tissue Culture Agar (Lab M Limited) was added and the medium was

autoclaved.

Different stress-inducing media were also used based on the standard ½ MS medium with a

solution of a substance added to them. Different media were prepared containing 25 and 100

mM mannitol (Sigma), 50 and 150 mM NaCl, 10 and 20 µM rotenone (Sigma), 1 and 2 mM

H2O2 , 50 and 100 nM Paraquat (Sigma) and 50 µM antimycin A (Sigma). For H2O2, Mannitol,

Paraquat and NaCl, ½ MS medium was used as control medium as these substances were

dissolved in water. For Antimycin A, ½ MS medium with 1 mL DMSO added was used as control

medium as this substance was dissolved in DMSO. For Rotenone, ½ MS medium with 1 mL

ethanol was used as control medium as this substance is dissolved in ethanol.

2. Methods

2.1. General methods

2.1.1. Seed sterilization

The seeds were packaged in Miracloth (Calbiochem) and 70% ethanol was added for two

minutes. A mixture containing 20 mL 12% NaOCl solution and 30 mL purified water was then

added for 13 minutes (The NaOCl solution should not be on the seeds for longer than 13

minutes to prevent damage to the seeds). The seed packages were then washed five times for

four minutes with sterile purified water. During each step the bottle was shaken to ensure

that the seeds got in contact with the different solutions and that no traces of NaOCl were left

after the washing steps.

2.1.2. Sowing the seeds

The seed packages were opened and using tweezers the seeds were spread out on the petri

dishes containing ½ MS medium for a total of 16 to 32 seeds per petri dish depending on the

experiment. The petri dishes were sealed with Micropore tape (3M) to avoid contamination

and evaporation while still letting some air through. The seeds were sown in three technical

and biological repeats. The petri dishes containing the seeds were subsequently vernalized at


60

4 °C for two days and placed in the growth chamber (21 °C). Every two days the petri dishes

were randomized to avoid positional effects on the growth of the plants.

2.2. Gene expression analysis of T-DNA insertion mutants

2.2.1. Seeds

The seeds of different T-DNA insertion lines for SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80

and INT90 and wild type seeds were used for the gene expression analysis (Table 3).

2.2.2. Harvesting the seedlings

The plants were grown on ½ MS medium for 10 days. Sixteen seedlings were harvested for

each line and for each repeat. The plants were harvested using tweezers and put into 2 mL

tubes and they were frozen immediately using liquid nitrogen.

2.2.3. RNA extraction

The plant tissue was homogenized using a shaker (RETSCH). Two metal balls (3 mm) were

added to the 2 mL tubes and the tissue was homogenized for 60 seconds at a frequency of 20

s-1 using cooled adaptors. After that TRIzol reagent (Life Technologies) was added to the plant

material. The reaction tubes were mixed and chloroform was added. The extraction of nucleic

acids involves adding an equal volume of phenol and chloroform to an aqueous solution of

homogenized plant tissue, mixing the two phases, and allowing the phases to separate by

centrifugation. Centrifugation of the mixture yields two phases: the lower organic phase and

the upper aqueous phase which contains the nucleic acids. The reaction tubes were mixed

and centrifuged and the upper aqueous phase containing RNA was transferred to a new 2 mL

tube containing 70% ethanol. After that, the RNeasy Mini Kit (Qiagen) purification kit was

followed according to the manufacturer’s instructions to purify the RNA from the samples.

RNA purification involves a DNase treatment step and for this step DNase (Promega) was used.


61

2.2.4. RNA to cDNA conversion

After the RNA was purified the RNA was converted to cDNA. The RNA concentration was

measured using the Nanodrop appliance (Thermo Scientific). After that, the iScript cDNA

synthesis Kit (Bio-Rad) was used according to the manufacturer’s instructions to convert the

1 µg RNA to cDNA for all samples.

2.2.5. Quantitative real-time PCR

The automated Janus pipetting robot (Perklin Elmer) was used to prepare the 384-well plate

used for the LightCycler 480 (Roche Diagnostics). This robot puts 2.5 μL of LightCycler 480

SYBRgreen I mastermix, 0.5 μL cDNA and 2 μL primermix (containing forward and reverse

primers for the genes of interest) in each well of the 384-well plate. The Janus pipetting robot

was set to do two technical repeats.

When the 384-well plate was prepared, it was sealed, centrifuged for 1 minute and placed in

the LightCycler 480 (Roche Diagnostics). The LyghtCycler was set to run the following protocol:

a pre-incubation step (10 minutes at 95°C), an amplification step of 45 cycles (10 seconds at

95°C, 15 seconds at 60°C, 15 seconds at 72°C), a melting curve step (5 minutes at 95°C, 1

minute at 65°C, with the temperature rising to 95°C at a rate of 4.8°C per second) and a cooling

step (10 minutes at 40°C).

2.2.6. Data analysis

The LightCycler data was analyzed using an internal program that is available on the PSB

intranet server. The output of this program was analyzed using Microsoft Excel.

2.3. Gene expression analysis under stress conditions

2.3.1. Seeds

The seeds of a wild type line were used for the gene expression analysis under stress

conditions.


62


The seeds were grown on ½ MS medium using EMD Millipore meshes (Miracloth). The meshes

were used to allow easy transfer of the plants between the different growth media. After 10

days the meshes were transferred to petri dishes containing the ½ MS medium supplemented

with stress-inducing substances. After 24 hours the plants were harvested using tweezers and

put into 2 mL tubes and they were frozen immediately using liquid nitrogen.

2.4. Ploidy analysis of T-DNA insertion mutants

2.4.1. Seeds

For the cellular analysis two wild type lines and four T-DNA insertion lines were selected from

Table 3 based on their expression levels for the gene of interest.

2.4.2. Harvesting the leaves

After 21 days the third leaf was harvested. For each line three leaves were harvested for each

biological repeat. The leaves were harvested using tweezers and were placed on a petri dish.

Nuclei Extraction Buffer (Partec) was added to the petri dish (200 µL) and the leaves were

chopped with a clean razor blade to homogenize the leaves. The mixture of plant material and

Nuclei Extraction buffer was then filtered using a green Celltrics filter (Partec). The Celltrics

filter was rinsed by first using water and then Nuclei Extraction Buffer. The filter was placed

on a 3.5 mL collection tube (Partec) containing 800 µL DAPI Staining Solution (Partec). The

mixture of plant material and Nuclei Extraction Buffer was pipetted on the filter and the flow

through was added to the DAPI staining solution.

2.4.3. Flow cytometry

To measure the nuclear DNA content the CyFlow ML flow cytometer (Partec) was used. This

flow cytometer is equipped with a UV laser that is able to detect fluorescence intensities of


63

DAPI stained nuclei. The Partec CyFlow ML flow cytometer was first rinsed using 1 mL Cleaning

Solution (Partec) followed by 1 mL of purified water at flow through speed 5. The sheath fluid

bottle was filled with purified water and the waste barrel was emptied. The collection tubes

were placed in the flow cytometer and a standard protocol for DAPI staining was run. The flow

through speed was adjusted during the run to reach around 100 counts per second.


The flow cytometer data was analyzed using the FCS Express software (De Novo Software) and

the output of this program was analyzed using Microsoft Excel.

2.5. Expression analysis of SWIB5

2.5.1. Seeds

For the localization analysis different pSWIB5::GFP::GUS lines were used (Table 5). The seeds

were obtained by transforming wild type plants with plasmids containing the constructs using

Agrobacterium transformation.

2.5.2. Transformation of Agrobacterium tumefaciens with the plasmids

The plasmids of the constructs were pipetted in a 2 mL tube (eppendorf) containing a solution

of Agrobacterium tumefaciens. Using electroporation cuvettes (Gene Pulser Cuvette, Bio-Rad)

and an electroporation appliance the plasmids of the constructs were transformed into the

Agrobacterium strains. After electroporation 1 mL YEB medium was added to the

Agrobacterium strains and the mixture was transferred to 12 mL tubes and incubated for 2

hours at 28 °C on a shaker. After two hours 200 mL was spread out on a petri dish containing

YEB medium with spectinomycin, gentamycin and rifampicin.


64

2.5.3. Transformation of Arabidopsis using Agrobacterium tumefaciens

Arabidopsis thaliana seeds were sown in soil and six weeks after the seeds germinated the

flower stems were cut off. The flower stems were cut off to increase the amount of flowers

that will grow on each plant. Two weeks later the plants were ready to be transformed with

the Agrobacterium tumefaciens strains that contain the plasmids. The Agrobacterium strains

were grown in 1 mL LB medium without antibiotics in 50 mL tubes (Corning Incorporated).

They were incubated for 9 hours at 28 °C on a shaker and another 10 mL of LB medium was

added. They were then incubated overnight at 28 °C on a shaker and the OD600 was checked

(it should be around 1.7). After that 40 mL purified water containing 10% sucrose and 0.05%

Silwet L-77 surfactant (Helena Chemical Co.) was added. Immediately after adding the solution

to the Agrobacterium strain the plants that were ready to be transformed were submerged in

the solution. The plants were then put back on a tray and were covered with barrier food wrap

(Sarogold). After 24 hours the barrier food wrap was removed, the plants were grown for six

weeks and the seeds were harvested.


The seedlings were harvested on day 5, 9 and 12. The seedlings were harvested using tweezers

and were subsequently put in a 24-well plate (Corning Incorporated). The seedlings were

fixated by adding heptane to them for 10 minutes. The heptane was removed and GUS buffer

(50 mM NaH2PO4, 10 mM β-mercaptoethanol, 1 mM EDTA and 0.1% Triton X-100) was added.

The seedlings with the GUS buffer were then incubated for 24 hours at 37 °C in the dark. The

GUS buffer was removed and the seedlings were then cleared using 100% ethanol (the ethanol

was refreshed once after 7 hours). The next day the 100% ethanol was replaced with lactic

acid and the seedlings were carefully put on a glass slide using tweezers.


The seedlings were imaged using a DMLB binocular (Leica) fitted with a camera.


65

2.6. Localization analysis of the SWIB-family domain proteins

2.6.1. Plasmids

For the localization analysis in Nicotiana benthamiana different p35S::GENE::GSGreen

plasmids were used for the SWIB1, SWIB2, SWIB3, SWIB4, SWIB6, INT80 and INT90 genes. Also

plasmids containing markers for the mitochondria and the chloroplasts and a plasmid with a

P19 anti-viral RNA silencing construct were used. The glycerol stocks of these plasmids in

Agrobacterium tumefaciens were already available.

2.6.2. Plant preparation

Some wild type Nicotiana benthamiana seeds were sown in a small pot containing wet soil

and grown for two weeks. The two week old seedlings were transferred to a big pot (2

seedlings per pot) containing wet soil.

2.6.3. Co-infiltration of Nicotiana benthamiana leaves

A 5 mL culture of the Agrobacterium tumefaciens glycerol stock was started in YEB with the

appropriate antibiotics. Two days later the OD600 was measured and the culture was diluted

resulting in a 2 mL culture with an OD600 of 1.5. The culture was centrifuged and the

supernatant was replaced by 2 mL Infiltration Buffer (for 100 mL: 1 mL MgCl2, 2 mL 0.5 M MES,

100 μL 0.1 M acetosyringone, 96.9 mL purified water). The culture with the Infiltration Buffer

was incubated for two hours at room temperature on a shaker. The infiltration mixture that

was used to infiltrate Nicotiana benthamiana leaves consists of 333 μL of each Agrobacterium

tumefaciens solution for a total of 1 mL. When the plants were four weeks old the leaves were

infiltrated with the mixture by making a small cut in the abaxial surface of the leaf and bringing

the mixture in the leaf using a 1 mL syringe. The infiltrated plants were taken out of the growth

chamber three to six days later. A piece of the leaf that was infiltrated was cut out using a

scalpel, placed on a glass slide with a drop of purified water on it and covered with a cover

slide.


66


A LSM510 laser scanning microscope (Zeiss) was used to image the leaves.

2.7. Interaction analysis of SWIB5

2.7.1. Seeds

For the interaction analysis two independent p35S::SWIB5::GSGreen lines were used. The

seeds were obtained by transforming wild type plants with plasmids containing the constructs

using Agrobacterium tumefaciens transformation.

2.7.2. Growing and harvesting the seedlings

For each line 150 mg of sterile seed was put into five Erlenmeyer flasks of 500 mL and 300 mL

½ MS medium was added. These were grown for 7 days in the growth chamber on a shaker.

The plant material was harvested using a vacuum filter and frozen immediately using liquid

nitrogen.

2.7.3. Protein extraction

The plant material (50 g) was grinded in liquid nitrogen with a mixer for 10 minutes and 100

mL Extraction Buffer (25 mM Tris-HCl buffer (pH 7.6), 15 mM MgCl2, 150 mM NaCl, 15 mM

pNO2PhePO4, 60 mM β-glycerophosphate, 0.1% NP 40, 0.1 mM Na3VO4, 1 mM NaF, 1 mM

PMSF, 1 μM E64, EDTA-free Ultra Complete tablet (1/10 mL), 0.1% Benzonase Nuclease

(Novagen) and 5% ethyleenglycol) was added. After that 0.1% Benzonase Nuclease (Novagen)

was added and the mixture was grinded for another 2 minutes. The mixture was incubated for

30 minutes on a rotating mixing device at 4 °C. The mixture was transferred to cooled

centrifugation tubes and centrifuged two times at 36900 x g for 20 minutes. To separate the

proteins from other compounds the supernatant was filtered through a double layer of mesh

(Miracloth).


67

2.7.4. Tandem affinity purification

In the first binding step GFP-Trap agarose beads (Chromotek) were transferred to a Poly-Prep

Chromatography Column (Bio-Rad) and Extraction Buffer was added. The protein extract was

added to the column and run through it at 1 mL per minute using a peristaltic pump at 4°C.

The column was then washed with Wash Buffer. In the first elution step the GFP-Trap agarose

beads were transferred to a 1.5 mL tube (Eppendorf) and Wash Buffer was added together

with HRV 3C Protease to cut off the first tag enzymatically. The mixture was incubated for 1

hour at 16°C on a rotating wheel and was then transferred to a Mobicol Column (Mobitec).

Wash Buffer was added to the column and the eluate was collected. In the second binding

step Streptavidin Sepharose High Performance beads (GE Healthcare Life Sciences) were used

and in the second elution step the second tag was cut off chemically using Desthiobiotin. The

proteins were concentrated using TCA precipitation and stored at -70 °C.


The identification of the interacting proteins of SWIB5 by mass spectrometry was outsourced

to an external lab.

2.8. Leaf size analysis


After 21 days the petri dishes with the seedlings on them were harvested. The seedlings were

taken out of the ½ MS medium using tweezers and the leaves, including the petioles, were cut

off at the stem of the seedling. The leaves were ordered according to their emergence time

(cotyledons, leaf one, leaf two, leaf three, etc.) and placed on square petri dishes (24 cm x 24

cm) containing 1% Plant Tissue Culture Agar. Cuts were made in the leaves to make sure they

were flat on the gel and they were placed with the adaxial surface facing up as this prevents

the leaves from curling up. For each line and condition ten seedlings were harvested for each

biological repeat.


68


Images were taken of the square petri dishes with the leaves on them using a camera. The

images of the petri dishes were taken with a light source behind them to reduce the

background noise and the same set-up was always used to take these images. The images

were imported in the image processing and analysis program ImageJ. Using three scripts that

are compatible with ImageJ the images were analysed. The first script converts the images to

black and white. These images are then cut up resulting in ten images per petri dish each

containing the leaves of one seedling using the second script. Using these images the third

script calculates the area of each leaf individually. The data was then analyzed in Microsoft

Excel and the statistical significance was determined using the statistical analysis software SAS

(SAS Institute Inc.).

2.9. Cellular analysis

2.9.1. Harvesting the leaves

The third leaf was harvested from 21 day old seedlings. For each line ten leaves were

harvested for each of the three biological repeats. The leaves were harvested using tweezers

and were subsequently put in a 24-well plate. The leaves were then cleared using 100%

ethanol (the ethanol was refreshed once after 7 hours). The next day the ethanol was replaced

with lactic acid and the leaves were carefully put on a glass slide using tweezers with the

abaxial surface facing up.

2.9.2. Making the cell drawings

Images were taken of each leaf to measure their area using ImageJ and the three leaves closest

to the average value were selected using Microsoft Excel. The three leaves that were selected

were then used to make the cell drawings. The cell drawings were made with a Jetstream

black ink pen (Mitsubishi Pencil Co.) using a DMLB microscope (Leica) fitted with a drawing

tube and a differential interference contrast objective. For each leaf 70-100 cells were drawn.

The same spot in the middle of the leaf was chosen for all leaves to avoid measuring

differences in cell size caused by choosing a spot closer or further to the leaf edge.


69


The cell drawings were scanned and analyzed using an internal program that is available on

the PSB intranet server. This program measures the cell number, the individual cell size and

the stomata number are measured for each leaf and the output of this program was analyzed

using Microsoft Excel.

2.10. Root analysis

2.10.1. Sowing the seeds

The seed packages were opened and using tweezers the seeds were placed on the petri dishes

containing ½ MS medium. On each petri dish six seeds were placed spread out on a line that

was drawn with a fiber-tip pen one centimeter from the side of the petri dish.


After 12 days the petri dishes were taken out of the growth chamber and they were scanned.

The images were imported in the image processing and analysis program ImageJ. The primary

root length, the lateral root length and the amount of lateral roots was measured Using

ImageJ. The data was analyzed using Microsoft Excel.

70

References

Andriankaja M, Dhondt S, De Bodt S, Vanhaeren H, Coppens F, De Milde L, Muhlenbock P, Skirycz A,

Gonzalez N, Beemster GT, Inze D (2012) Exit from proliferation during leaf development in Arabidopsis thaliana:

a not-so-gradual process. Dev Cell 22: 64-78

Autran D, Jonak C, Belcram K, Beemster GT, Kronenberger J, Grandjean O, Inze D, Traas J (2002) Cell

numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene. EMBO J 21:

6036-6049

Barkan A (2011) Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold.

Plant Physiol 155: 1520-1532

Bennett-Lovsey R, Hart SE, Shirai H, Mizuguchi K (2002) The SWIB and the MDM2 domains are

homologous and share a common fold. Bioinformatics 18: 626-630

Beyer P (2010) Golden Rice and 'Golden' crops for human nutrition. N Biotechnol 27: 478-481

Colas des Francs-Small C, Falcon de Longevialle A, Li Y, Lowe E, Tanz SK, Smith C, Bevan MW, Small I

(2014) The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are

Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I. Plant

Physiol 165: 1409-1416

De Veylder L, Larkin JC, Schnittger A (2011) Molecular control and function of endoreplication in

development and physiology. Trends Plant Sci 16: 624-634

Edgerton MD (2009) Increasing crop productivity to meet global needs for feed, food, and fuel. Plant

Physiol 149: 7-13

Eloy NB, de Freitas Lima M, Van Damme D, Vanhaeren H, Gonzalez N, De Milde L, Hemerly AS, Beemster

GT, Inze D, Ferreira PC (2011) The APC/C subunit 10 plays an essential role in cell proliferation during leaf

development. Plant J 68: 351-363

71

Eloy NB, Gonzalez N, Van Leene J, Maleux K, Vanhaeren H, De Milde L, Dhondt S, Vercruysse L, Witters

E, Mercier R, Cromer L, Beemster GT, Remaut H, Van Montagu MC, De Jaeger G, Ferreira PC, Inze D (2012)

SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early development and

A-type cyclin stabilization. Proc Natl Acad Sci U S A 109: 13853-13858

Foley JA, Defries R, Asner GP, Barford C, Bonan G, Carpenter SR, Chapin FS, Coe MT, Daily GC, Gibbs HK,

Helkowski JH, Holloway T, Howard EA, Kucharik CJ, Monfreda C, Patz JA, Prentice IC, Ramankutty N, Snyder PK

(2005) Global consequences of land use. Science 309: 570-574

Gonzalez N, Inze D (2015) Molecular systems governing leaf growth: from genes to networks. J Exp Bot

66: 1045-1054

Gonzalez N, Vanhaeren H, Inze D (2012) Leaf size control: complex coordination of cell division and

expansion. Trends Plant Sci 17: 332-340

Gonzali S, Loreti E, Solfanelli C, Novi G, Alpi A, Perata P (2006) Identification of sugar-modulated genes

and evidence for in vivo sugar sensing in Arabidopsis. J Plant Res 119: 115-123

Govindaraj M, Vetriventhan M, Srinivasan M (2015) Importance of genetic diversity assessment in crop

plants and its recent advances: an overview of its analytical perspectives. Genet Res Int 2015: 431487

Hedtke B, Wagner I, Borner T, Hess WR (1999) Inter-organellar crosstalk in higher plants: impaired

chloroplast development affects mitochondrial gene and transcript levels. Plant J 19: 635-643

Hisanaga T, Kawade K, Tsukaya H (2015) Compensation: a key to clarifying the organ-level regulation of

lateral organ size in plants. J Exp Bot 66: 1055-1063

Hudik E, Yoshioka Y, Domenichini S, Bourge M, Soubigout-Taconnat L, Mazubert C, Yi D, Bujaldon S,

Hayashi H, De Veylder L, Bergounioux C, Benhamed M, Raynaud C (2014) Chloroplast dysfunction causes multiple

defects in cell cycle progression in the Arabidopsis crumpled leaf mutant. Plant Physiol 166: 152-167

72

Koch MS, Ward JM, Levine SL, Baum JA, Vicini JL, Hammond BG (2015) The food and environmental

safety of Bt crops. Front Plant Sci 6: 283

Kondorosi E, Roudier F, Gendreau E (2000) Plant cell-size control: growing by ploidy? Curr Opin Plant

Biol 3: 488-492

Liao JC, Hsieh WY, Tseng CC, Hsieh MH (2015) Dysfunctional chloroplasts up-regulate the expression of

mitochondrial genes in Arabidopsis seedlings. Photosynth Res

Liu W, Stewart CN, Jr. (2015) Plant synthetic biology. Trends Plant Sci 20: 309-317

Marshall A, Aalen RB, Audenaert D, Beeckman T, Broadley MR, Butenko MA, Cano-Delgado AI, de Vries

S, Dresselhaus T, Felix G, Graham NS, Foulkes J, Granier C, Greb T, Grossniklaus U, Hammond JP, Heidstra R,

Hodgman C, Hothorn M, Inze D, Ostergaard L, Russinova E, Simon R, Skirycz A, Stahl Y, Zipfel C, De Smet I (2012)

Tackling drought stress: receptor-like kinases present new approaches. Plant Cell 24: 2262-2278

Melonek J, Matros A, Trosch M, Mock HP, Krupinska K (2012) The core of chloroplast nucleoids contains

architectural SWIB domain proteins. Plant Cell 24: 3060-3073

Ray S, Satya P (2014) Next generation sequencing technologies for next generation plant breeding. Front

Plant Sci 5: 367

Rogers C, Oldroyd GE (2014) Synthetic biology approaches to engineering the nitrogen symbiosis in

cereals. J Exp Bot 65: 1939-1946

Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of

oxidative stress on Arabidopsis mitochondria. Plant J 32: 891-904

Szabala BM, Osipowski P, Malepszy S (2014) Transgenic crops: the present state and new ways of genetic

modification. J Appl Genet 55: 287-294

Taira Y, Okegawa Y, Sugimoto K, Abe M, Miyoshi H, Shikanai T (2013) Antimycin A-like molecules inhibit

cyclic electron transport around photosystem I in ruptured chloroplasts. FEBS Open Bio 3: 406-410

73

Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327:

818-822

Tian YC, Qin XF, Xu BY, Li TY, Fang RX, Mang KQ, Li WG, Fu WJ, Li YP, Zhang SF, et al. (1991) Insect

resistance of transgenic tobacco plants expressing delta-endotoxin gene of Bacillus thuringiensis. Chin J

Biotechnol 7: 1-13

Tominaga-Wada R, Ishida T, Wada T (2011) New insights into the mechanism of development of

Arabidopsis root hairs and trichomes. Int Rev Cell Mol Biol 286: 67-106

White DW (2006) PEAPOD regulates lamina size and curvature in Arabidopsis. Proc Natl Acad Sci U S A

103: 13238-13243

Witzany G (2006) Serial Endosymbiotic Theory (set): the biosemiotic update. Acta Biotheor 54: 103-117

Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (beta-

carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287: 303-305

74

Attachments

Attachment 1: Leave size analysis on stress

75

Attachment 2: Protocols

MS Medium

Medium recepten zijn voor 1L medium. 1L medium volstaat om plus minus 10 platen

petriplaten te vullen. (100 mL per plaat)

- 1L nanopure water

- 2,15 g Murashige & Skoog medium

- 0,5 g MES

- Breng pH op 5,8 met 1M KOH oplossing

- 10 g Plant Tissue Culture Agar

- Voeg de oplossing hieraan toe

- Autoklaveer het medium

Zaaien

- Zaaien op medium

o Platen worden met een steriele tandenstoker vlak onder het

mediumoppervlakte gezaaid in de flow.

o Elke plaat bevat meerdere lijnen die in gelijke proporties worden uitgezaaid.

Een controle lijn wordt steeds meegenomen.

o Platen worden met micropore tape afgeplakt om te sterieliteit te vrijwaren

- Zaaien op mesh

o Wanneer transfer naar nieuw medium op een bepaalde dag vereist is, wordt

gezaaid op steriele nylon mesh die over het gestolde medium wordt geplaatst.

Het is belangrijk om de luchtbellen tussen de mediumlaag en de mesh te

verwijderen.

o Zaaien gebeurt eveneens met een steriele tandenstoker in de flow.

o Transfer van de mesh gebeurt op de gekozen dag met 2 steriele pincetten.

o Elke plaat bevat meerdere lijnen die in gelijke proporties worden uitgezaaid.

Een controle lijn wordt steeds meegenomen.

o Platen worden met micropore tape afgeplakt om te sterieliteit te vrijwaren

76

Groei

- Planten werden gegroeid geurende 22 dagen bij 21°C met licht van 6u tot 22u.

Rozetanalyse

- Haal op de 22e dag na stratisficatie de platen uit de groeikamer en fotografeer de

platen.

- Fotografeer ook een meetlat die zal worden gebruikt worden om de schaal in te stellen

voor de analyse van de rozetoppervlakte.

- Open de foto van de schaal in de ImageJ software. Stel de schaal in zoals in de

rozetanalyse tijdens tijdens het in soil experiment.

- Open de foto’s van de platen in ImageJ en maak deze zwart wit. (process, binary, make

binary).

- Meet de rozetoppervlakte door met de “wand” in ImageJ elke plant afzonderlijk aan

te klikken en meet de oppervlakte via de sneltoets ‘m’.

- Exporteer de resultaten naar Excel om de gemiddelde rozetoppervlakte per lijn en per

conditie te berekenen.

Cellulaire analyse

- Oogst van elke plant van het experiment blad nummer 3, en ontkleur deze in 100%

alcohol.

- Wanneer de bladeren ontkleurd zijn, vervang de alcohol door lactic acid om de

bladstructuur te versoepelen.

- Leg de bladeren op microscopieplaatjes met de adaxiale kant naar onder… kant naar

onder, voeg een druppel lactic acid toe, en dek af met een dekglaasje.

- Fotografeer een schaal en de bladeren met behulp van de LEICA binoculair en het NIC

elements (NIKON) software programma.

- Open de ImageJ software en open de foto van de schaal om de schaal in te stellen.

77

- Open vervolgens de foto’s van de balderen, en bereken de oppervlakte door met de

penseel de omtrek van het blad te overtekenen, en op de sneltoets ‘m’ te drukken om

de oppervlakte te meten.

- Exporteer de meetresultaten naar Excel, en bereken de gemiddelde oppervlakte van

blad 3 per lijn en per conditie.

- Kies 3 bladeren uit per lijn en per conditie met een oppervlakte die dicht aanleunt

tegen het gemiddelde.

- Teken de abaxiale cellen van deze bladeren met behulp van de LEICA DIC microscoop

op een 40X vergroting, met behulp van de tekentubus.

- Sluit de cellen op celtekeningen zodat er geen openingen in de lijnen meer aanwezig

zijn.

- Scan de tekeningen in.

- Open de ImageJ software en stel de schaal van 40X in.

- Open de ingescande celtekeningen in ImageJ. Maak zwart/wit (process, binary, make

binary) en maak de lijnen dikker (process, binary, dilate).

- Stel de meetinstelling juist in zodat de meetresultaten worden weergegeven met 6

cijfers na de komma. (analyse, set measurements)

- Meet de oppervlakte van de cellen met behulp van de “wand” en met behulp van de

‘m’ sneltoets. Elke gemeten cel wordt ingekleurd in zwart, zodat cellen niet meermaals

worden gemeten. (stomata worden niet gemeten)

- Exporteer de meetdata naar Excel.

- Bereken de gemiddelde celoppervlakte, en aan de hand van deze resultaten eveneens

het aantal cellen per blad.

qRT-PCR

- Koel een 384-well plaat af op een stuk aluminum folie op ijs

- Prepareer mastermixen die de primers en SybrGreen bevatten (met behulp van Janus

robot)

- Uitendelijk bevat elke well:

2μl 0,5μM Primers (Forward en Reverse tesamen)

78

3μl SybrGreen Mastermix I (Roche)

1μl cDNA

- Dek de plaat af met aluminium film

- Centrifugeer kort

- Plaats plaat in LightCycler480

- Open LightCycler480 Software en log in

- Start nieuw experiment

- Gebruik volgende template:

Pre-incubatie: 1 cyclus (10 minuten op 95°C)

Amplificatie: 45 cycli (10sec op 95°C, 15sec op 60°C, 15sec op 72°C)

Smelt curve: 1 cyclus (5 minuten op 95°C, 1 minuut op 65°C, tot 95°C aan

4,8°C/sec)

79

Afkoeling: 1 cyclus (10 minuten op 40°C)

Data analyse

- Controleer of er slechts1 Tm peak aanwezig is:

In LightCycler480 Software, klik analysis

Kies Tm Calling

Klik calculate

In elke well zou er steeds slechts 1 Tm peak mogen zijn (=1 specifiek qPCR

product)

Gebruik geen stalen die meer dan 1 Tm piek hebben voor verdere analyse.

- Exporteer de Ct waarden naar Excel:

In LightCycler480 Software, klik op analysis

Kies 2nd Derivative Max

Klik calculate

In de table met de Cp-waarden, klik op rechter muisknop en kies Export Table

Sla op al seen tekst bestand (tab-delimited)

- In Excel, open het tab-delimited tekst bestand

- Voor verdere analyse te vergemakkelijken: herschik de data zodat de originele plaats

wordt weergegeven.

- Alle volgende stappen zijn per staal (per genotype, per behandeling,…)

- Bereken het gemiddelde van de 2 housekeeping genes

- Trek dit gemiddelde af van de Ct waarden van het staal: dit is de ΔCt waarde.

- Trek de ΔCt waarde af van de behandeling van interesse van de of the treatment ΔCt

van de control behandeling (ΔCt Control – ΔCt Treatment): dit is de ΔΔCt waarde.

- Positieve en negatieve ΔΔCt waarden wijzen op resp. up- and down-regulatie

vergeleken met het controle staal.

80

Plant TAP Protocols:

Buffer composition:

Extraction buffer:

25mM Tris/HCl pH 7.6, 15mM MgCl2, 150mM NaCl, 15mM pNO2Phenyl PO4, 60mM β-

glycerophosphate, 0.1% NP-40, 0.1mM Na3VO4, 1mM NaF, 1mM PMSF, 1μM E64, EDTA-free

Ultra Complete tablet (1/10mL), 0.1% benzonase, 5% ethyleenglycol

Wash buffer:

10mM Tris/HCl pH 7.6, 150mM NaCl, 0.1% NP-40, 0.5mM EDTA, 1μM E64, 1mM PMSF, 5%

ethyleenglycol

Streptavidin elution buffer:


ethyleenglycol, 20mM desthiobiotine

Extraction:

- grind 50g harvested plant material in liquid N2 with kitchen blender for 10 minutes and

evaporate liquid N2

- add 100mL extraction buffer

- add 0.1% Benzonase Nuclease(Novagen) and mix for another 2 minutes

- incubate 30 minutes on a rotating mixing device at 4°C

- transfer mixture to prechilled centrifugation tubes and centrifuge two times at 36900g

for 20 minutes

- filter supernatant through a double layer of miracloth mesh (22-25µm)

- determine protein concentration via Bradford method

Plant GSTEV and GSrhino TAP protocol:

1. Binding and Washing Step1 (IgG)

- equilibrate 100μL(1CV) = 133 μL slurry(75%) IgG-Separose 6 Fast Flow (GE Healthcare)

in extraction buffer (3x10CV) and transfer to polyprep column (Biorad)

81

- remove bottom of 50mL Falcon with Stanley knife and assemble on polyprep column.

Seal with parafilm

- load total protein extract stepwise on assembled IgG-Sepharose column at

1mL/minute with peristaltic pump in cold room at 4°C

- wash with 15mL (150CV) wash buffer

2. Elution Step1 (TEV or Rhinovirus 3C Protease)

- transfer IgG-Sepharose beads to 1.5mL Eppendorf tube, add 400μL wash buffer and

100U AcTEV or Rhinovirus 3C protease

- incubate for 1h at 16 °C for TEV or 4°C for Rhinovirus 3C protease on a rotating wheel.

After 30 minutes, give an additional boost of 100U protease

- collect eluate by passing on a Mobicol column and wash beads with 400µl wash buffer

3. Binding and Washing Step2 (Streptavidin)

- incubate eluate on 100μL(1CV) = 200ul slurry(50%) in wash buffer equilibrated

Streptavidin Sepharose High Performance (GE Healthcare) for 1h at 4°C on a rotating

wheel in a 1,5mL protein lo-bind eppendorf tube

- transfer mix to polyprep column and wash with 10 mL TEV(100CV) buffer on a polyprep

column(Bio-Rad)

4. Elution Step2 (1xSB+Desthiobiotin)

- apply 1 mL Streptavidin elution buffer to column, incubate 5 minutes and collect eluate

by gravity

- concentrate proteins by TCA precipitation(25%)

- store eluate(pellet) at -70°C

Plant GSgreen and GSyellow TEV/Rhino TAP protocol:

1.Binding and Washing Step1 (GFP-trap agarose)

- equilibrate 100μL(1CV) = 200 μL slurry(50%) GFP-trap agarose (Chromotek) in

extraction buffer (3x10CV) and transfer to polyprep column (Biorad)

82

- remove bottom of 50mL Falcon with Stanley knife and assemble on polyprep column.

Seal with parafilm

- load total protein extract stepwise on assembled IgG-Sepharose column at

1mL/minute with peristaltic pump in cold room at 4°C

- wash with 15mL (150CV) wash buffer

2.Elution Step1 (TEV or Rhinovirus 3C Protease)

- transfer GFP-trap agarose beads to 1.5mL Eppendorf tube, add 400μL wash buffer and

100U AcTEV or Rhinovirus 3C protease

- incubate for 1h at 16 °C for TEV or 4°C for Rhinovirus 3C protease on a rotating wheel.

After 30 minutes, give an additional boost of 100U protease

- collect eluate by passing on a Mobicol column and wash beads with 400µl wash buffer

3.Binding and Washing Step2 (Streptavidin)

- incubate eluate on 100μL(1CV) = 200ul slurry(50%) in wash buffer equilibrated

Streptavidin Sepharose High Performance (GE Healthcare) for 1h at 4°C on a rotating

wheel in a 1,5mL protein lo-bind eppendorf tube

- transfer mix to polyprep column and wash with 10 mL TEV(100CV) buffer on a polyprep

column(Bio-Rad)

4.Elution Step2 (1xSB+Desthiobiotin)

- apply 1 mL Streptavidin elution buffer to column, incubate 5 minutes and collect eluate

by gravity

- concentrate proteins by TCA precipitation(25%)

- store eluate(pellet) at -70°C

I.One-step GFP Purification protocols

Buffer composition:

Extraction buffer:

83

25mM Tris/HCl pH 7.6, 15mM MgCl2, 150mM NaCl, 15mM pNO2Phenyl PO4, 60mM β-

glycerophosphate, 0.1% NP-40, 0.1mM Na3VO4, 1mM NaF, 1mM PMSF, 1μM E64, EDTA-free

Ultra Complete tablet (1/10mL), 0.1% benzonase, 5% ethyleenglycol

Wash buffer:


ethyleenglycol

Extraction:

- grind 15g harvested plant material in liquid N2 with kitchen blender for 10 minutes and

evaporate liquid N2

- add 30mL extraction buffer

- add 0.1% Benzonase Nuclease(Novagen) and mix for another 2 minutes

- incubate 30 minutes on a rotating mixing device at 4°C

- transfer mixture to prechilled centrifugation tubes and centrifuge two times at 36900g

for 20 minutes

- filter supernatant through a double layer of miracloth mesh (22-25µm)

- determine protein concentration via Bradford method

GFP Purification:

- input of 100mg on 30μL (1CV) =60μL slurry(50%) in extraction buffer equilibrated

(3x10CV) GFP-trap Agarose (GTA Chromotek) in a 50mL Falcon

- incubate 1h at 4°C on a rotating mixing device

- centrifuge for 5min at 450g and remove unbound fraction

- transfer beads to Mobicol column

- wash with 6mL wash buffer

- elute with 40µL 1xNuPAGE sample buffer (Invitrogen) at 70°C with regular mixing

- add 9 volumes of icecold ethanol to precipitate proteins and incubate overnight at -

70°C

- centrifuge at 13000g for 20min at 4°C

- remove ethanol and air dry the sample pellet

- dissolve pellet in 30µL 1xNuPAGE sample buffer and heat for 10min at 70°C

Date post:	11-Sep-2019
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Characterization of new putative leaf growth regulators...

Documents