C H A P T E R T H I R T Y

M

IS

*{

ethods

SN 0

DepaDepa

Bioimaging Techniques for

Subcellular Localization of Plant

Hemoglobins and Measurement of

Hemoglobin-Dependent Nitric Oxide

Scavenging In Planta

Kim H. Hebelstrup,* Erik �stergaard-Jensen,† and Robert D. Hill*

Contents

1. In

in

076

rtmrtme

troduction

Enzymology, Volume 437 # 200

-6879, DOI: 10.1016/S0076-6879(07)37030-4 All r

ent of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canadant of Molecular Biology, University of Aarhus, Aarhus, Denmark

5

8 Elsevie

ights rese

96

2. M

easuring Hemoglobin-Dependent NO Scavenging 5 96

3. T

echniques for Determination of Subcellular Localization of Plant

Hemoglobins

597

4. Im

aging of Hemoglobin-Dependent NO Scavenging in Arabidopsis

Plants

598

5. E

ngineering of GLB1-GFP/GLB2-GFP Constructs and Microscopic

Analysis of A. thaliana Plants Expressing GFP-Tagged Hemoglobin

600

Refe

rences 6 03

Abstract

Plant hemoglobins are ubiquitous in all plant families. They are expressed at

low levels in specific tissues. Several studies have established that plant

hemoglobins are scavengers of nitric oxide (NO) and that varying the endoge-

nous level of hemoglobin in plant cells negatively modulates bioactivity of NO

generated under hypoxic conditions or during cellular signaling. Earlier meth-

ods for determination of hemoglobin-dependent scavenging in planta were

based on measuring activity in whole plants or organs. Plant hemoglobins do

not contain specific organelle localization signals; however, earlier reports on

plant hemoglobin have demonstrated either cytosolic or nuclear localization,

depending on the method or cell type investigated. We have developed two

bioimaging techniques: one for visualization of hemoglobin-catalyzed scaveng-

ing of NO in specific cells and another for visualization of subcellular

r Inc.

rved.

595

596 Kim H. Hebelstrup et al.

localization of green fluorescent protein-tagged plant hemoglobins in trans-

formed Arabidopsis thaliana plants.

1. Introduction

Hemoglobin genes are ubiquitous among plants, and various experi-ments have indicated that their primary role is to scavenge nitric oxide(NO) (Dordas et al., 2003; Perazzolli et al., 2004). This function has beensuggested to play an important role during hypoxic situations, particularly inroots (Igamberdiev and Hill, 2004), but it is also involved with modulatingNO signaling during development in normoxic shoots (Hebelstrup et al.,2006). Because hemoglobin is an effective NO-scavenging agent in plants,cellular localization of hemoglobin is of interest. The high reactivity and theshort lifetime of NO in a cell environment limit the distribution of NOfrom its source. Tissue and even intracellular levels may, therefore, beexpected to vary considerably. Initial experiments used to determine cellu-lar localization of plant hemoglobins have presented contradictory resultsdepending on the method and model plant used.

This chapter describes two bioimaging techniques used for the localiza-tion of hemoglobins and NO in plant tissue: (A) detection of tissue- and/orcell-specific NO levels altered by overexpression or silencing of hemoglo-bin expression in Arabidopsis thaliana and (B) imaging of subcellular locali-zation of plant hemoglobins using transgenic A. thaliana plants expressinggreen fluorescent protein (GFP)-tagged plant hemoglobin.

Arabidopsis thaliana contains three classes of hemoglobin genes (Trevaskiset al., 1997; Watts et al., 2001). Hemoglobin-dependent scavenging of nitricoxide was studied by comparing NO levels in wild-type plants with lineshaving overexpression of either class 1 hemoglobin Glb1 or class 2 hemo-globinGlb2 (35S:Glb1 or 35S:Glb2) or with silencing (Hg:Glb1) or knock-out (Glb2dSpm) of the genes. The engineering of lines has been describedelsewhere (Hebelstrup et al., 2006). Engineering of constructs for A. thalianalines with expression of GFP-tagged Glb1 or Glb2 is described here.

2. Measuring Hemoglobin-Dependent

NO Scavenging

Electron paramagnetic resonance spectroscopy (Dordas et al., 2003)and chemiluminescent detection of NO in emission gases from leaves(Perazzolli et al., 2004) have been used previously for the measurement ofhemoglobin-dependent NO scavenging in planta. These methods are usefulfor analytical determination of hemoglobin-dependent scavenging of NO

Bioimaging Techniques for Subcellular Localization 597

in a whole organ or even a whole plant. Under some circumstances, such asthe involvement of NO in cell signaling, a method that is tissue or cellspecific may be preferred. The fluorescent probe 4,5-diaminofluoresceindiacetate (DAF-2 DA) has been used to visualize and determine the tissue-and cell-specific presence of nitric oxide in plant tissues in several studies.NO levels were determined, for example, in stomatal guard cells loadedwith DAF-2 DA to show that NO is a central component in the signaltransduction of abscisic acid-induced stomatal closure (Desikan et al., 2002;Garcia-Mata and Lamattina, 2002). DAF-2 DA has also been used tovisualize NO during the gravitropic response of horizontally placed rootsof Pisum sativum (Hu et al., 2005), showing that NO accumulates nonsym-metrically in a similar fashion to the hormone auxin. DAF-2 DA was usedto confirm low NO levels in root and stomatal guard cells of plants with agenetically based impairment of NO synthesis (Guo et al., 2003). Weprovide here a technique to image cellular and subcellular hemoglobin-dependent nitric oxide scavenging in specific tissues using the NO-specificfluorescent probe DAF-2 DA. We observed that overexpression of planthemoglobin effectively scavenges nitric oxide, even when present at highlevels. We, therefore, suggest that modulating cellular levels of hemoglobincan be used together with DAF-2 DA to confirm when NO is formed inspecific cells and is involved in physiological processes.

3. Techniques for Determination of

Subcellular Localization of Plant

Hemoglobins

No plant hemoglobin genes have been reported to contain nuclearlocalization signals and, therefore, subcellular localization of plant hemo-globins to the cytosol would be expected. In line with this, hemoglobin ispresent in the cytosolic fraction of cell extracts of alfalfa root cells(Igamberdiev et al., 2004). However, when using different localizationtechniques and/or different plant species, nuclear localization hasbeen observed. Electron microscopy of immunogold-labeled hemoglobinin cultured alfalfa root cells indicated a strong tendency to nuclear localiza-tion (Seregelyes et al., 2000). Similarly, it has been reported that cottonhemoglobin, tagged with GFP, shows a tendency to nuclear localizationwhen expressed transgenically in onion epidermal cells (Qu et al., 2005).This discrepancy in reported subcellular localization of plant hemoglobinsindicates that localization is dynamic and depends on cell type or conditionof the cell studied. Since the first report on genetic GFP tagging of specificproteins and its use in studying expression and localization (Chalfie et al.,1994), GFP and derived fluorescent proteins have been used widely in many

598 Kim H. Hebelstrup et al.

cell types and organisms, including plants (Chudakov et al., 2005). Thetechnique reported here uses GFP-tagged hemoglobin in a whole organism,using a nontransgenic strategy, offering a method for imaging native, taggedhemoglobins in live A. thaliana cells under various conditions.

4. Imaging of Hemoglobin-Dependent NO

Scavenging in Arabidopsis Plants

We use the NO-activated fluorescent probe DAF-2 DA for detectionof NO in A. thaliana cells. Plant organs are loaded with DAF-2 DA andprepared for microscopy as described later. The incubation time used givessatisfactory fluorescent levels with A. thaliana leaves and inflorescences.Wounding activates the formation of NO in plant tissue, and strong NOlevels are detected around wounds such as the petiole or stem where theorgan has been cut from the plant. It is therefore not recommended to cutorgans close to the cells of interest. Fluorescence is detected well with eithera mercury-lamp fluorescent microscope or a laser-scanning confocalmicroscope. Fluorescence microscopy is carried out with a Zeiss Axioplan2 microscope. Filter settings are as follows: excitation window, 460–480 nm;emission window, 505–530 nm. When using these filter settings, autofluor-escence is nearly undetectable in untreated A. thaliana epidermal and rootcells. The following protocol is used for loading DAF:

1. The plant organ is incubated for 2 h in DAF loading buffer:10 mMMES-Tris (pH 5.6), 0.1 mM CaCl2, and 10 mM KCl containing either(a) 200 mM cPTIO (Calbiochem,Merck KGaA, Darmstadt, Germany), aspecific scavenger of NO to provide reference samples without NO, or(b) 0.4 mM sodium nitroprusside (SNP; Sigma-Aldrich Denmark A/S,Denmark) or an equivalent NO donor molecule.

2. DAF-2 DA is added to a final concentration of 10 mM in the DAFloading buffer and incubation is continued for 1 h. DAF-2 DA willreact specifically with intracellular NO because DAF-2 DA is onlyactivated for reaction with NO after modification by intracellularesterases.

3. Organs are washed briefly in DAF loading buffer and are then ready formicroscopy.

Figure 30.1 shows NO-dependent fluorescence of DAF-2 DA in floralbuds and young flowers visualized by fluorescence microscopy. Very littleNO is detected in wild-type A. thaliana young flowers (see Figs. 30.1C and30.1D). However, in young flowers and floral buds from plants withsilencing of Glb1 (Hg:Glb1), a higher level of nitric oxide is detected (seeFigs. 30.1A and 30.1B). Negative controls of Glb1-silenced plants with the

Figure 30.1 Detection of NO-activated fluorescence of DAF-2DA inA. thaliana flow-erswith fluorescencemicroscopy.WhenGlb1 is silenced, a high endogenous nitric oxideconcentration is observed in various organs, including floral buds and young flowers.(A) Transmissionwhite light image of young flowers from plant with silencing ofGlb1.(B) Fluorescence image of same flowers as in A. (C)Transmissionwhite light image of ayoungwild-typeA. thaliana flower. (D) Fluorescence image of the same flower as in C.

Bioimaging Techniques for Subcellular Localization 599

addition of cPTIO show a fluorescence level close to wild-type lines,confirming that NO is the component responsible for activation of DAF-2 DA fluorescence. Positive controls of wild-type plants with the additionof 1.2 mM SNP have a very high fluorescence level. Interestingly, over-expression of either Glb1 or Glb2 in 35S:Glb1 or 35S:Glb2 lines preventsaccumulation of intracellular NO generated from 1.2 mM SNP, demon-strating that ectopic expression of plant hemoglobins can scavenge highlevels of intracellular nitric oxide. Hb overexpression may, therefore, beuseful in examining situations in which NO effects are difficult to detect.When hemoglobin is overexpressed, NO is scavenged and its effect shoulddisappear. This method has the advantage that NO scavenging can bedirected to specific tissues, cells, or situations by choosing a promoter thatwill be active only in specific cells or under certain conditions. We also usedlaser-scanning confocal microscopy for detection of nitric oxide-dependent

600 Kim H. Hebelstrup et al.

activation of DAF-2 DA with a Zeiss LSM 510 META microscope. Thesamples are excited by an argon laser at 488 nm. NO-dependent DAF-2 DAfluorescence is detected in a 505- to 530-nm window. Chlorophyllautofluorescence is detected in a 670- to 690-nm window. Figure 30.2shows a three-dimensional confocal image of how NO accumulates in thehydathode of a Glb1-silenced (Hg:Glb1) A. thaliana leaf. Gene expressionstudies have shown that hemoglobin is expressed specifically at hydathodesin A. thaliana leaves (Hebelstrup et al., 2006) and, in line with this, no NOaccumulation is detected in the hydathodes of wild-type A. thaliana leaves.

5. Engineering of GLB1-GFP/GLB2-GFP

Constructs and Microscopic Analysis of

A. thaliana Plants Expressing GFP-Tagged

Hemoglobin

Constructs for GFP tagging of A. thaliana hemoglobin gene classes1 and 2 (Glb1 and Glb2) are prepared by first purifying total RNA fromseedlings of A. thaliana (Col-0) with a commercial kit (RNeasy Plant MiniKit, Qiagen) according to the manufacturer’s instructions. cDNA is con-structed with M-MLV reverse transcriptase (Promega, WI) using oligo(dT)primers (Qiagen). Glb1 and Glb2 open reading frames are amplified bypolymerase chain reaction (PCR) from the cDNA using the followingprimers: 50-TCTAGAGGTTGTGAAATATTATGGAG-30 and 50-TCTAGAGTTGGAAAGATTCATTTCAG-30 for Glb1 and 50-TCTAGATGGGAGAGATTGGGTTTACA-30 and 50-TCTAGACTCTTCTTGTTTCATCTCGG-30 for Glb2. PCR fragments are ligated into the vector pCR-TOPO and cloned in Escherichia coli using a commercial kit (TOPO TAcloning kit, Invitrogen) according to the manufacturer’s description. pCR-TOPO plasmids containing clonedGlb1 orGlb2 are digested withXbaI, andfragments are ligated into theXbaI site in the T-DNA region of the modifiedTi plasmid pPZP211–35S-GFP-pANOS (Anderssen et al., 2005) to generatethe two constructs 35S::GLB1-GFP and 35S::GLB2-GFP. These constructscontain the open reading frame of either Glb1 or Glb2 followed by a GFP-coding sequence, giving rise to C-terminal-tagged hemoglobin whenexpressed in a plant host. Constructs also contain a selection marker forkanamycin resistance and should be useful for transformation methods,including direct DNA bombardment and transformation of plant tissuewith Agrobacterium tumefaciens. We tested the constructs in A. thaliana byfirst cloning the constructs in E. coli (DH10) and then subsequently inA. tumefaciens for eventual transformation of A. thaliana plants by the floraldipmethod (Clough and Bent, 1998). Positive first-generation transformants(T1) are selected by growth in medium containing 50 mg/ml kanamycin.

Figure 30.2 Three-dimensional projection of confocal scanningmicroscopic imagingof an enlarged hydathode from aGlb1-silenced plant. An elevatedNO level is found in aspherical region consisting of chlorophyll-less cells at the tip of the hydathode. Chloro-phyll (A) and NO (B) were detected as described in the text. (C) A merged image is ofA andB.

Bioimaging Techniques for Subcellular Localization 601

Figure 30.3 Confocal image of roots cells of transgenicA. thaliana plants with ectopicexpression of GFP-tagged hemoglobins Glb2-GFP (A) by the 35S:Glb1-GFP or Glb2-GFP (B) by the 35S:Glb2-GFP constructs. Both types of hemoglobin localize through-out the cytosol and nucleoplasm of the cells. However, a higher concentration may beseen in the nucleus.

602 Kim H. Hebelstrup et al.

Bioimaging Techniques for Subcellular Localization 603

Expression of GFP-tagged hemoglobin in T2 seedlings is confirmed byexamination with both fluorescence and confocal microscopy. In epidermalcells, strong fluorescence is detected in stomatal guard cells. In roots, fluores-cence is found in all cell types. GLB1-GFP and GLB2-GFP show similarlocalization patterns. Fluorescence is observed throughout the cytosol andnucleus; however, it is absent in the nucleolus (Fig. 30.3). There appears to bea higher concentration in the nucleus than in the cytosol. Lines with variouslevels of fluorescence are examined, and the subcellular distribution pattern issimilar in all lines.

In summary, this chapter presented two bioimaging techniques:oneshowing that hemoglobin-catalyzed scavenging of NO can be altered inplanta by the modulation of intracellular hemoglobin levels and anotherdemonstrating that GFP-tagged hemoglobins can be used for the visualiza-tion of subcellular localization of plant hemoglobins. These techniquespresent resources for studying the effect of NO metabolism in plant cellsand for studying further the function of plant hemoglobins.

REFERENCES

Anderssen, S. U., Cvitanich, C., Gronlund, M., Busk, H., Jensen, D. B., and Jensen, E. O.(2005). Vectors for reverse genetics and expression analysis. In ‘‘Lotus Japonicus Hand-book’’ (A. J. Marquez, ed.). Kluwer Academic, New York.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994). Greenfluorescent protein as a marker for gene expression. Science 263, 802–805.

Chudakov, D. M., Lukyanov, S., and Lukyanov, K. A. (2005). Fluorescent proteins as atoolkit for in vivo imaging. Trends Biotechnol. 12, 605–613.

Clough, S. J., and Bent, A. F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.

Desikan, R., Griffiths, R., Hancock, J., and Neill, S. (2002). A new role for an old enzyme:Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-inducedstomatal closure in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 99, 16314–16318.

Dordas, C., Hasinoff, B. B., Igamberdiev, A. U., Manac’h, N., Rivoal, J., and Hill, R. D.(2003). Expression of a stress-induced hemoglobin affects NO levels produced by alfalfaroot cultures under hypoxic stress. Plant J. 35, 763–770.

Garcia-Mata, C., and Lamattina, L. (2002). Nitric oxide and abscisic acid cross talk in guardcells. Plant Physiol. 128, 790–792.

Guo, F. Q., Okamoto, M., and Crawford, N. M. (2003). Identification of a plant nitricoxide synthase gene involved in hormonal signaling. Science 302, 100–103.

Hebelstrup, K. H., Hunt, P., Dennis, E., Jensen, S. B., and Jensen, E. O. (2006). Hemoglo-bin is essential for normal growth of Arabidopsis organs. Physiol. Plant. 127, 157–166.

Hu, X., Neill, S. J., Tang, Z., and Cai, W. (2005). Nitric oxide mediates gravitropic bendingin soybean roots. Plant Physiol. 137, 663–670.

Igamberdiev, A. U., and Hill, R. D. (2004). Nitrate, NO and haemoglobin in plantadaptation to hypoxia:An alternative to classic fermentation pathways. J. Exp. Bot. 55,2473–2482.

604 Kim H. Hebelstrup et al.

Igamberdiev, A. U., Seregelyes, C., and Manac’h, N., and Hill, R. D. (2004). NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglo-bin. Planta 219, 95–102.

Perazzolli, M., Dominici, P., Romero-Puertas, M. C., Zago, E., Zeier, J., Sonoda, M.,Lamb, C., and Delledonne, M. (2004). Arabidopsis nonsymbiotic hemoglobin AHb1modulates nitric oxide bioactivity. Plant Cell 16, 2785–2794.

Qu, Z. L., Wang, H. Y., and Xia, G. X. (2005). GhHb1:A nonsymbiotic hemoglobin geneof cotton responsive to infection by Verticillium dahliae. Biochim. Biophys. Acta Gene Struct.Express. 1730, 103–113.

Seregelyes, C., Mustardy, L., Ayaydin, F., Sass, L., Kovacs, L., Endre, G., Lukacs, N.,Kovacs, I., Vass, I., Kiss, G. B., Horvath, G. V., and Dudits, D. (2000). Nuclearlocalization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfacells. FEBS Lett. 482, 125–130.

Trevaskis, B., Watts, R. A., Andersson, C. R., Llewellyn, D. J., Hargrove, M. S.,Olson, J. S., Dennis, E. S., and Peacock, W. J. (1997). Two hemoglobin genes inArabidopsis thaliana:The evolutionary origins of leghemoglobins. Proc. Natl. Acad. Sci.USA 94, 12230–12234.

Watts, R. A., Hunt, P. W., Hvitved, A. N., Hargrove, M. S., Peacock, W. J., andDennis, E. S. (2001). A hemoglobin from plants homologous to truncated hemoglobinsof microorganisms. Proc. Natl. Acad. Sci. USA 98, 10119–10124.