Molecular basis of Plant resistance to Abiotic stresses like High temperature

and Heavy metals

Introduction

• Higher plants are sessile and therefore cannot escape abiotic stress factors

• This immobile nature requires more protection and this enabled them cope with the different stress factors

• Thus the plants have to alter their physiologies, metabolic mechanisms, gene expressions and developmental activities to cope with the stress effects

• Gene products play a key role in the molecular mechanisms of stress tolerance in plants.

Different types of Abiotic stress

• Water stress• Salt stress• High temperature stress• Freezing stress• Photo-oxidative stress• Nutrient stress• Heavy metal stress

High Temperature stress

• High temperature stress in plants arises in response to many factors such as :

exposure of plants to high ambient temperatures exposure of germinating seeds to the soil which is

warmed by absorbed infrared radiation from the sun more plant transpiration followed by less water

absorption reduced transpiration capacity in certain plant organs forest fires natural gas blowouts, etc.

Effect of Heat stress

• Membranes self assemble from amphipathic lipids as a result of hydrophobic interactions. Therefore, their properties will depend critically on temperature.

• Membrane components suited to one temperature will be inappropriate at other temperatures.

• Both membranes and proteins need to be flexible in order to function in biological systems; this restricts the temperature range over which they can be biologically active.

Heat shock proteins• Because of temperature sensitivity of the forces responsible for protein

folding , proteins are easily denatured by high temperature.

• Biological organisms have a suite of proteins that are made in response to high temperature that appear to be designed to prevent or reverse the effects of heat on protein denaturation called heat shock proteins (HSPs) which are not present, or are present in small quantities in unstressed organisms.

• These HSPs are involved in cellular repair, rescue, cleanup and/or protection during the stress and from its recovery.

• The hsps are divided into families based on their molecular weight, eg. Hsp 100 (with mol. Wt. between 100 to 104)

• The different hsp families appear to have different functions.

Different families of Hsp• Hsp 100• Hsp 90• Hsp 70 • Hsp 60

• heat stress may affect membrane function and protein folding

• Two plant processes that are particularly sensitive to heat stress are pollen development and photosynthesis.

• Recent evidence indicates that there are heat shock factors with DNA binding domains that may be important components in the transduction pathway between high temperature stress and gene expression leading to accumulation of heat shock proteins

miRNA

• Wheat miRNAs showed differential expression in response to heat stress by using Solexa high-throughput sequencing cloned the small RNAs from wheat leaves treated by heat stress

• Among the 32 miRNA families detected in wheat, nine conserved miRNAs were putatively heat responsive.

• For example: miR172 was significantly decreased, and miRNAs

(including miR156, miR159, miR160, miR166, miR168, miR169, miR393 and miR827) were upregulated under heat stress

siRNA

• Abiotic stress responsiveness has also been observed in a pool of T. aestivum small noncoding RNAs

• In wheat seedlings heat stress substantially change the expression of four siRNAs:

siRNA002061_0636_3054.1 is strongly downregulated by heat

siRNA 005047_0654_1904.1 is greatly downregulated by heat

siRNA080621_1340_0098.1 is downregulated by heat

Strategies for improving heat tolerance

• Improving heat tolerance has been attempted by various biotechnological methods:

Controlling the composition of membranes. For example, Arabidopisis plants can be engineered to prevent them from making trienoic fatty acids in their chloroplasts.

Improving heat stress is engineering constitutive or over-expression of Hsps. Hsp 100 family members and the smhsps have both been implicated in thermotolerance of plants

Perhaps more promise lies in overexpressing heat shock factors. These are genes that control the expression of other genes and so changing the expression of one heat shock factor will affect the expression of many genes that through evolution, have come to be controlled by the same control.

Heavy metal stress• It is apparent that complex network of transport, chelation, and

sequestration processes has evolved over time that functions in maintaining concentrations of essential metal ions in different cellular compartments within a narrow physiological range, thus minimizing the damage caused by entry of non-essential metal ions into the cytosol

• Important components of heavy metal homeostasis and detoxification systems are:

membrane-based heavy metal transporters intracellular metal chaperones for efficient distribution of

scarce essential metals, chelation and sequestration processes

• Loss of any one of these critical processes will lead to hypersensitivity to heavy metal ions.

4 main families of metal transport proteins

• P-type ATPases; e.g.heavy-metal ATPases (P1B)

• Cation diffusion facilitators (CDF-transporters)

• ZRT-/IRT- like proteins (ZIP-transporters)

• Natural resistance associated Macrophage proteins (Nramp-transporters); e.g. AtNramp

Iron-regulated zinc-regulated transporters

• Arabidopsis IRT1 gene (first isolated transporter gene) is a major transporter responsible for high affinity iron uptake from the soil

• Its abundance is controlled at both levels of transcript and protein accumulation. Overexpression of IRT1 does not confer dominant gain-of-function enhancement of metal uptake.

Iron deficiency results in induction of IRT1 transcript accumulation Iron sufficiency results in reduction of IRT1 transcript levels. • High levels of zinc and cadmium also contribute to reduction in IRT1

transcript levels. • ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, thus

suggesting roles in transport of zinc from the soil into the plant. • ZIP4 is induced in both shoots and roots of zinc-limited plants, thus it may be

involved in the transport of zinc either intracellularly or between plant tissues.

Chelation• Once metal ions enter the cell, they are bound by chelators and

chaperones. Chelators contribute to metal detoxification by buffering cytosolic metal

concentrations Chaperones specifically deliver metal ions to organelles and metal-

requiring proteins.

• Among heavy metal- binding ligands in plant cells are: Phytochelatins (PCs) : Overexpression of the wheat PCS gene TaPCS1 in

Nicotiana glauca (shrub tobacco) also increased tolerance to metals such as Pb and Cd.

Metallothioneins (MTs): In Saccharomyces cerevisiae cup1∆ mutant ABDE-1 (metal-sensitive) confirmed the functional nature of this mcMT1 genein sequestering both essential (Cu, Zn) and non-essential metals (Cd, Pb, Cr).

Chaperones• In recent years a family of soluble metal receptor proteins,

known as “metallochaperones”, that are active in intercellular metal trafficking has emerged.

• For example: In prokaryotic cells and Arabidopsis thaliana, small cytosolic

proteins, designated as copper chaperones, have been identified.

A copper chaperone (CCH) and a responsive to antagonist 1 (RAN1), both identified in A. thaliana, were the first Cu delivery systems identified in plant cells

Orthologues of the three copper chaperones characterized in yeast, ATX1, CCS and COX17, have been found in A. thaliana

miRNA

• miRNAs are involved in plant responses to nutrient stress• According to recent studies with Arabidopsis, miR399, miR395,

and miR398 are induced in response to phosphate-, sulfate-, and copper-deprived conditions, respectively.

• Phosphate homeostasis is partially controlled through miR399, which targets a gene encoding a putative ubiquitin- conjugating enzyme (UBC24).

• miR399 is upregulated in low phosphate-stressed plants and is not induced by other common stresses, whereas its target, UBC24 mRNA, was reduced primarily in roots of plants exposed to low-phosphate stress

Phosphate• It is likely that an MYB transcription factor, PHOSPHATE STARVATION

RESPONSE 1 (PHR1), is involved in miR399 expression. PHR1 is expressed in response to phosphate starvation and positively regulates a group of phosphate-responsive genes by binding to GNATATNC cis-elements [78–80]. This cis-element has been found upstream of all known miR399 genes in Arabidopsis [76,78]. Further- more, phr1 mutants show a significant decline in miR399 induction under phosphate stress [76,78]. miR399 has been isolated from phloem, and its abundance in phloem increases upon phosphate starvation

• miRNAs themselves can be subject to posttranscrip- tional regulation, as revealed by the discovery of the INDUCED BY PHOSPHATE STARVATION1 (IPS1) gene, which acts as a target mimic to control miR399 action

Sulfur

• Sulfur (S) is one of the essential macronutrients and is available in the form of sulfate in the soil.

• miR395 targets both ATP sulfurylases (APSs) and the sulfate transporter AST68.

• Sulfate deprivation induces the expression of miR395 with a concomitant decrease in transcript levels of APS1.

• The abundance of miR395 in the phloem increases for Brassica plants deprived of S, and the increase was much stronger in the phloem than in the root, stem, or leaf tissue

Copper• The plant micronutrient copper (Cu) is essential for photosynthesis,

oxidative responses, and other physiological processes.

• miR398 is a key regulator of Cu homeostasis.

• When Cu is limiting, the level of miR398 increases, and this reduces the allocation of Cu to CSDs (CSD1 and CSD2) and therefore makes Cu available for other essential processes.

• In Brassica under Cu deprivation, miR398 is upregulated not only in leaf, stem, and root tissue but also in phloem sap.

• According to more recent research, Brassica phloem sap contains a specific set of small RNAs that are distinct from those in leaves and roots, and the phloem responds specifically to stress

• In higher plants, the Cu/Zn-SODs are replaced by Fe-SODs

Cadmium• Cadmium (Cd) is one of the most toxic metals in agricultural soils.

• Recent studies identified a set of conserved and non-conserved miRNAs that are differentially regulated in response to heavy metals in rice.

• miRNAs help regulate plant responses to heavy metal stress in addition to other abiotic stresses. In B. napus, expression of miRNAs shows different responses to sulfate deficiency and Cd exposure

• miR160 was transcriptionally downregulated by sulphate deficiency and by Cd exposure

• miR164b and miR394a,b,c in roots and stems were upregulated by sulfate deficiency

• Similarly, treatment with Cd induced expression of miR164b and miR394a,b,c in all tissues except that miR164b was downregulated in leaves.

• miR156a and miR167b in roots and miR156a and miR167c in leaves were upregulated under sulfate deficiency. In contrast, miR167a and miR168 in roots and miR167a, miR167b, and miR168 in leaves were downregulated.

• Under Cd stress, most B. napus miRNAs are induced. Notably, miR156a, miR167a, and miR167c in roots and miR167a and miR167c in leaves were strongly upregulated.

• Rice miRNAs showed different patterns of expression in leaves and roots. miR601, miR602, and miR603 in roots were upregulated while miR602 and miR606 in leaves and miR604 in roots were downregulated by Cd exposure

Mercury and Aluminium• Mercury (Hg) and aluminum (Al) regulate the expression of miRNAs

in M. truncatula.

• miR171, miR319, miR393, and miR529 are upregulated in response to Hg, Cd, and Al

• miR319 showed weak constitutive expression in leaves. It is upregulated by Cd and Al but was not affected by Hg.

• Similarly, expression of miR393 was not affected by Al but was slightly upregulated by Hg and Cd.

• In contrast, miR166 and miR398 are downregulated by Hg, Cd, and Al exposure

Iron

• Expression of miRNAs also is affected by Fe deficiency, in Arabidopsis

miR169b, miR169c, miR172c, miR172d, miR173 and miR394b in roots

miR169c, miR172c, miR172d, miR173, miR394a and miR394b in shoots

initially upregulated and then down- regulated during the period of Fe deficiency.

• These results indicate that the cloned miRNAs respond to Fe deficiency

Thank-you…

By:Nazish Nehal,M. Tech (Biotechnology),University School of Biotechnology (USBT),Guru Gobind Singh Indraprastha University,New Delhi (INDIA)