Role of Chloroplast Heatshock Proteins Involved in Abiotic Stress Tolerance in Wheat

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Chloroplastic heatshock proteins also functions in maturing the wheat grains and thus improves the flour quality. These heat shock proteins basically assist and adjust the folding aggregations of storage proteins during the process of grain filling. The HSPs synthesis during embryogenesis in the developing embryos conferred the protection of seeds from the heat stress (Chauhan et al. 2012). Chlorplast Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle (Inoue et al. 2013).

An agrobacterium-mediated transformation of chloroplast localized HSP gene, Oshsp26, from Oryza sativa to Festuca arundinacea ( a type of grass) conferred the heat tolerance, oxidative tolerance. An increase in photosystem II efficiency in transgenic varieties have been observed (Kim et al. 2012). Similarly, agrobacterium-mediated transformation of a Cp-HSP gene, LeHSP21, from Solanum lycopersicum was constitutively expressed in Nicotiana tabacum, driven by the cauliflower mosaic virus 35S promoter. The transgenic varieties revealed enhanced tolerance to both high temperature and oxidative stress. Moreover, it also improved and increased the activities of anti-oxidants such as proline (Zhang et al. 2016).

Heat shock proteins also shown to induce the thermotolerance in bent grass Agrostis stolonifera. In creeping bent grass, an increase in photochemical efficiency of photosystem II (Fv/Fm) was measured during heat stress due to production of heat shock proteins, and decrease in shoot electrolyte leakage (ShEL), and hiher root viability (RV) were monitored (Wang et al. 2014).

HSP101 was shown to be a major component of thermotolerance in Arabidopsis (Maestri et al. 2002). Additionally, Hsp10 as co-chaperonins have been identified from plants such as Oryza sativa, Hordeum vulgare, Triticum aestivum, Phaseolus vulgaris and Pisum sativum (Nitnavare et al. 2016). Chloroplastic heat shock protein had been reported to protect the plant’s photosynthesis and electron transport chain during heavy metal stress from Nickel and Pb like harmful metals (Heckathorn et al. 2004).

Another abundant class of Heat shock proteins, HSP90 has also protective role in response to stress. Identification and isolation of 12 genes of GmHsp90s from Glycine max. (soybean) and Quantitative real-time PCR of these genes revealed their experssion and function under abiotic stress. The PCR data revealed that genes induced under heat, osmotic and salt stress but not cold stress. The clonong and overexpression of the 5 GmHsp90s in Arabidopsis thaliana provided evidences that GmHsp90s can reduce the damages caused by the abiotic stress (Xu et al. 2013).

Chloroplast heatshock proteins have also been shown to induce tolerance against oxidative stress. Expression of Hsp21(the nuclear encoded chloroplast heat shock protein) in Arabidopsis thaliana revealed that transgenic plants are more resistant to oxidative stress and heat stress due to over expression of Hsp. Enhanced Hsp synthesis acts as ROS scavenging and induces tolerance against oxidative stress (Härndahl et al. 1999) .

To date it is identified and revealed that chloroplast small heat shock proteins have the variety of roles such as protection of seed development, increasing efficiency of PSII, protection of protein complexes of thylakoid membrane and protein stabilization and protein transport as well. These roles have been elucidated during heat stress, heavy metal stress and oxidative stress as well.

There is strong and evident need to elucidate the molecular basis of salt and drought tolerance in wheat. The aim of this study is to identify beneficial chloroplast HSP genes and to utilize them in molecular breeding programmes with a target to produce transgenic salt and drought tolerant varieties of wheat in the future.

OBJECTIVES:

- To identify the chloroplast HSPs in wheat.

- To understand the role of CHSPs in abiotic stress tolerance in wheat.

- To investigate the effect of Cp-HSP on photosystem-II.

METHODOLOGY:

Wheat is one of the oldest and most important of the cereal crops. Wheat is a C3 plant and belongs to poaceae family. Wheat is a widely grown crop. It is grown from temperate, irrigated to dry and high-rain-fall areas and from warm, humid to dry, cold environments.. The minimum water content required in the grain for wheat germination is 35 to 45% by weight. Germination may occur between 4° and 37°C, optimal temperature being from 12° to 25°C (Acevedo et al. 2002).

- Wheat Germplasm collection: collection of wheat seeds.

Seed size does not alter germination but affects growth, development and yield. Bigger seeds have several advantages when compared to smaller seeds, such as faster seedling growth, higher number of fertile tillers per plant and higher grain yield. The advantage of bigger seeds is demonstrated when the crop is grown under environmental stresses, particularly drought (Acevedo et al. 2002).

- Germination and plant growth. Plants would be grown in different pots. For control samples, frequent watering will be done to some pots.

- Salt stress application: Different concentrations of salt (150, 200, 400, 600, and 800 mM) would be used to water the plants instead of distilled water (Haq et al. 2013).

- Morphological observations.

- Physiological investigation:

- Measurment of chlorphyll content (Hiscox and Israelstam 1979).

- Superoxide dismutase activity (Beauchamp and Fridovich 1971).

- Measurment of Peroxidase activity (Gorin and Heidema 1976).

- Photosystem II efficiency: chlorophyll fluorescence (Fv'/Fm') measurement by (Wang et al. 2008).

- Sampling: In sampling leaves will be collected from plant growing in normal conditions (control samples) and from the plants growing under stress conditions.

- DNA isolation by CTAB method from T.aestivum leaves (Porebski et al. 1997; Doyle 1987).

- RNA isolation by TRIZOL reagent (Chomczynski and Sacchi 1987).

- Retrieving the sequences of CpHSP from database.

- Primer designing for normal PCR and RT-PCR.

- cDNA synthesis fom RNA by