Stress Tolerance Variety In Medicinal Plant

4.29 ± 0.09 b

3.92 ± 0.08 a


ns nonsignificant, significant at the **0.01, and ***0.001 levels of probability, respectively; for each Triticum species, different letters in the same column refer to significant differences between genotypes. Source: Dias and Lidon (2009)

ns nonsignificant, significant at the **0.01, and ***0.001 levels of probability, respectively; for each Triticum species, different letters in the same column refer to significant differences between genotypes. Source: Dias and Lidon (2009)

growth and net assimilation rates of shoot (Wahid 2007). Likewise, heat shock affected the meristematic activity and reduced the growth of various parts mainly the leaves (Salah and Tardieu 1996) . Applied heat stress arrested the cell wall elongation and altered cell differentiation (Potters et al. 2007) .

Reproductive growth is more critically affected by the prevailing high temperature stress during anthesis and seed growth. Pollination is especially sensitive to heat stress. The mature pollens are more sensitive, and quite often fail to fertilize (Dupuis and Dumas 1990) . Heat stress interferes with the development of pollen mother cell and microspore and causes male sterility (Sakata et al. 2000; Sato et al. 2006; Abiko et al. 2005). In the event of successful pollination, heat stress affected the kernel development in maize (Monjardino et al. 2005) and reduced the kernel density and reproductive growth in maize, wheat and Suneca during kernel development (Wilhelm et al. 1999; Maestri et al. 2002).

Dias and Lidon (2009) did not find any effect of heat stress on number of grains per spike in both durum and bread wheat; nonetheless upon exposure to heat stress during grain growth, grain size was substantially reduced in both bread and durum wheats (Table 6.1). Likewise, heat stress also reduced the grain yield in both wheats (Table 6.1). However, different genotypes responded variably in terms of grain size and grain yield and a strong relationship between genotypes and temperature has been observed (Table 6.1).

High temperature also results in the boll and flower bud abortion in cotton, pea, and brassica, possibly owing to limited water supply and nutrients during reproductive development (Hall 1992; Guilioni et al. 1997; Young et al. 2004). During seed development, heat stress was found to affect seed storage process and kernel quality like starch and protein metabolism during grain filling in maize (Wilhelm et al. 1999; Maestri et al. 2002) .

Plant Heat Acclimation

Fig. 6.1 Hypocotyl elongation phenotype of different pea varieties for 36 h in the dark at 22°C after heat acclimation and stress [(a) local variety, (b) Shandong variety, (c) Taiwan variety]. Left is expressed as control (22°C); middle is expressed as the germinated seeds were acclimated at 37°C for 1 h, followed at 22°C for 1 h, and then stressed at 48°C for 2 h. Right is expressed as the seeds were stressed at 48°C for 2 h. After Tian et al. (2009) with permission

Fig. 6.1 Hypocotyl elongation phenotype of different pea varieties for 36 h in the dark at 22°C after heat acclimation and stress [(a) local variety, (b) Shandong variety, (c) Taiwan variety]. Left is expressed as control (22°C); middle is expressed as the germinated seeds were acclimated at 37°C for 1 h, followed at 22°C for 1 h, and then stressed at 48°C for 2 h. Right is expressed as the seeds were stressed at 48°C for 2 h. After Tian et al. (2009) with permission

2.2 Anatomical and Developmental Responses

Like other abiotic stresses, heat stress brings about quite a few morphogenetic and histological modifications. At whole plant level, generally cell size is reduced (Santarius 1973; Berry and Bjorkman 1980). There may be several mor-pho-anatomical modifications in cells and tissues such as increased densities of stomata and trichomes and greater xylem vessels area of shoot and root in Lotus creticus seedlings (Banon et al. 2004). On exposure of grapes to heat stress, cell membrane permeability was substantially increased and mesophyll cells were severely damaged (Zhang et al. 2005). High temperature also causes various changes at subcellular level. For instance, in chloroplast, it changed the thyla-koids structure in maize (Karim et al. 1997) and resulted in loss of swelling and stacking of grana (Gounaris et al. 1984). In grapes, heat stress damaged the mesophyll cells, which showed round-shaped chloroplasts, swollen stroma lamellae, badly affected the antenna complex of photosystem (PS) II (Carpentier 1999), clump formation of vacuolar contents, disrupted cristae and deformed mitochondria (Zhang et al. 2005).

Heat stress restricted the emergence and elongation of hypocotyls in three pea (Pisum sativum) varieties (Tian et al. 2009). Nonetheless, heat acclimation for 1 h at 37°C improved the germination and hypocotyl development (Fig. 6.1). In the sprouting buds of sugarcane, heat stress badly affected the differentiation of various cells and tissues (Rasheed 2009). Here the major changes were noted on mesophyll cell expansion and development of vascular connections (Fig. 6.2).

2.3 Physiological and Metabolic Responses

In hot environments, plants exhibit various physiological and metabolic responses. The most

Fig. 6.2 Sprouting bud of sugarcane under control condition (left). Effect of heat stress on the histological changes in sprouting buds of sugarcane after 36 h of exposure (middle) and role of proline in mitigating heat stress effect (right). Source: Rasheed (2009)

Fig. 6.2 Sprouting bud of sugarcane under control condition (left). Effect of heat stress on the histological changes in sprouting buds of sugarcane after 36 h of exposure (middle) and role of proline in mitigating heat stress effect (right). Source: Rasheed (2009)

important of those may be the changes in carbon fixation, oxidative stress; tissue water status and metabolites accumulation. These responses are briefly discussed below.

2.3.1 Photosynthesis

Heat stress causes photosynthetic acclimation and alters the physiological processes directly and changes the developmental patterns indirectly (Downton and Slatyer 1972). All the steps, processes, and aspects of photosynthesis are prone to increased ambient temperature (Al-Khatib and Paulsen 1990). The photosynthesis in C3 plants is more affected by high temperature than C4 plants (Wahid and Rasul 2005). The maize seedling grown at 25°C and transferred to 35°C for 20 min led to 50% inhibition in photosynthesis (Sinsawat et al. 2004). Maize showed maximum net photosynthesis near 31°C, decreased at temperature above 37°C and was completely inhibited near 45°C (Crafts-brandner and Salvucci 2002) . Heat stress diminished the net photosyn-thetic (Pn) and stomatal conductance substantially in many plant species (Ranney and Peet 1994; Crafts-Brandner and Salvucci 2002; Morales et al. 2003); in this regard, Pn in developed leaves was more sensitive than mature leaves (Karim et al. 1997, 1999).

Photosynthetic apparatus are highly sensitive to heat temperature and inhibited when leaf temperature exceed 38°C in most plants (Edwards and Walker 1983). PS-II, water splitting and oxygen evolving complex (OEC) in photosynthesis are more heat-sensitive components of photosynthesis (Havaux 1993; Pastenes and Horton 1996a; Heckathorn et al. 1998a). Extensive studies show that both PS-I and PS-II are damaged by increased temperature. In barley and potato, heat stress damaged PS-I and PS-II and affected electron transport (Havaux 1998; Szilvia et al. 2005). Heat stress damaged the antenna complex of PS-II and reduced photosynthetic behavior (Carpentier 1999; Rokka et al. 2000; Zhang et al. 2005). High temperature during greening led to the inactivation of PS-I and PS-II (Sasmita and Narendranath 2002). High temperature increased chlorophyll a:b ratio and decreased chlorophyll:carotenoid ratio in sugarcane (Wahid 2007) .

High temperature alters the energy sharing by changing the action of Calvin cycle and other metabolic processes such as photorespiration, synthesis and stability of the Rubisco enzyme (Holaday et al. 1992; Pastenes and Horton 1996b), disruption of electron transport activity and bound RUBP supply by heat stress (Ferrar et al. 1989) . Extreme temperature reduced the activation state of Rubisco enzyme in the exposed leaf tissue and increased the RUBP (Feller et al. 1998; Crafts-Brandner and Law 2000), which inhibited photosynthesis as compared to control plants (Sharkey et al. 2001). High temperature enhances chlorophyllase activity and decreases the quantities of photosynthetic pigments (Todorov et al. 2003). The loss of chlorophyll is good indicator of heat tolerance in wheat (Ristic et al. 2007, 2008) . High temperature modifies the activities of carbon metabolism enzymes, especially the Rubisco (Ferrar et al. 1989; Holaday et al. 1992; Pastenes and Horton 1996a, b). Moreover, activities of starch and sucrose synthesis enzymes are greatly influenced (Chaitanya et al. 2001; Vu et al. 2001).

2.3.2 Reactive Oxygen Species and Oxidative Damage

Like other abiotic stresses, heat stress evokes the ROS generation including hydrogen peroxide (H2O2), superoxide radical (O2-), singlet oxygen ('O2) and hydroxyl radical (OH) , and induces oxidative stress (Mittler 2002; Taiz and Zeiger 2006; Potters et al. 2007). Chloroplast and mitochondria are the major sites where superoxide radicals are regularly produced, whereas some quantities are also produced in microbodies. Principally, ROS causes peroxidation of membrane lipids, destruction of pigments, and modification of membrane functions (Xu et al. 2006). The OH- appears to be more damaging than other ROS, which is formed with the combination of O2- and H2O2 in the presence of Fe2+ and Fe3+ in trace amounts in Haber-Weiss reaction (Apel and Hirt 2004) . The OH- is greatly damaging to chlorophyll, proteins, lipids, DNA, and other important macromolecules (Sairam and Tyagi 2004).

Tolerant plants have the tendency to protect themselves from the damaging effects of ROS with the synthesis of various antioxidant systems (Apel and Hirt 2004). This protection starts with the conversion of O2 - by superoxide dismutase (SOD) into H2O2 with the help of ascorbate per-oxidase (APX) or catalase (CAT). A number of physiological processes are affected by the overexpression of SOD in plants, including removal of H2O2; toxic reductants, biosynthesis and degradation of lignin in cell walls, auxin catabolism, etc. (Scandalios 1993). Activation of APX is due to the physiological injuries occurring in plants under heat stress (Mazorra et al. 2002).

Increased levels of ROS under high temperature cause cellular injury due to reduced antioxi-dant activity in the stressed tissues (Fadzillah et al. 1996; Mittler et al. 2004). In order to increase the heat tolerance, the levels and activities of antioxidants must be increased to protect against high temperature-induced oxidative stress. Studies conducted in this regard revealed that heat acclimated turf grass showed reduced ROS production owing to enhanced ascorbate and glutathione synthesis (Xu et al. 2006). It is suggested that antioxidant capacity of cells can be increased by some signaling molecules (Gong et al. 1997; Dat et al. 1998). Nonetheless, research is imperative to add to the list of potential signaling molecules, which may enhance the antioxidant production in cells exposed to heat temperature stress (Wahid et al. 2007) .

2.3.3 Water Relations

Heat stress drives the rapid loss of water from the plant surface, causes tissue and organ dehydration, and restricts growth in plant species, for example, sorghum (Machado and Paulsen 2001), tomato (Mazorra et al. 2002), and sugarcane (Wahid and Close 2007). Heat stress produces osmotic strain on the growing tissues due to diminished root hydraulic conductance and tissue water status (Jiang and Huang 2001; Morales et al. 2003). Likewise, it may result in substantial reduction in sorghum (Sorghum bicolor) leaf growth and leaf water content and water potential in wheat (Shah and Paulsen 2003).

Heat stress also disrupts the uptake and translocation of water, ions, and organic solutes across the plant membranes, interferes with photosynthesis and respiration, increases evapo-transpiration rate, reduces the leaf osmotic potential and increases the chlorophyll fluorescence (Tsukaguchi et al. 2003; Huve et al. 2005; Taiz and Zeiger 2006). It results in stomatal closure and reduces the tissue water contents (Berry and Bjorkman 1980; Wahid et al. 2007). Heat stress-induced water stress thus is closely associated with reduction of soil water contents (Talwar et al. 1999).

2.3.4 Osmolytes Accumulation

Accumulation of certain low molecular mass organic compounds, generally called compatible solutes or osmoprotectants, is an important adaptive mechanism in plants subjected to abiotic stresses including temperature extremes (Hare et al. 1998; Sakamoto et al. 1998). Several osmolytes, including sugars and sugar alcohols (polyols), proline, tertiary, and quaternary ammonium compounds and tertiary sulphonium compounds, are reported to accumulate in different plant species exposed to stress conditions (Sairam and Tyagi 2004; Wahid et al. 2007).

Among different compatible solutes, enhanced synthesis of soluble sugars, free proline and glycinebetaine (GB) has been more frequently studied for their osmoregulatory and protective roles (Matysik et al. 2002; Bohnert et al. 2006; Wahid 2007; Wahid et al. 2008; Farooq et al. 2008a). GB plays a great role as osmoprotectant in plants under a range of abiotic stresses including high temperature (Sakamoto and Murata 2002). However, the ability of plants to synthesize GB under stressful conditions varies among species (Ashraf and Foolad 2007) . For example, sugarcane under heat stress (Wahid and Close 2007) and maize under drought (Quan et al. 2004) and chilling (Farooq et al. 2008c) and rice under drought (Farooq et al. 2008a) are reported to accumulate large amounts of GB. Like GB, increased free proline accumulation in higher plants in response to abiotic stresses has also been reported (Kavi Kishore et al. 2005). Biosynthesis of GB or proline may buffer the cellular redox potential under heat and other abiotic stresses, suggesting their functional significance (Rontein et al. 2002). The accumulation of soluble sugars was greatly implicated for improved heat tolerance of sugarcane (Wahid and Close 2007). In view of the importance of osmoprotectants accumulation, more concerted efforts on engineering pathways for enhanced biosynthesis of osmolytes may be fruitful (Ashraf and Foolad 2007).

2.3.5 Metabolite Synthesis

Heat stress leads to the accumulation of a range of primary and secondary metabolites. Primary metabolites are either direct products of carbon fixation (e.g., sugars, organic acids) or are synthesized after preliminary transformations of primary metabolites (e.g., amino acids, betaines alcohol sugars). Like other abiotic stresses, the accumulation of primary metabolites under heat stress has also been well documented (Iba 2002; Zhu 2003). Important primary metabolites showing accumulation under heat stress include free proline, GB, soluble sugars, etc., (Wahid 2007;

Wahid and Close 2007; Wahid et al. 2008). In a recent study, using cluster and principal component analyses, it was revealed that out of 122 primary and secondary metabolites determined using advanced techniques like GC-MS and amino acids analyzer, only sucrose, quinate, trans-aconitate, guanine, g-amino butyric acid (GABA), and ethanolamine held relationships with the high temperature tolerance of sugarcane bud chips (Rasheed 2009) .

On the contrary, the synthesis and accumulation of secondary metabolites are less well understood under high temperature stress. Secondary metabolites are biosynthesized in plants from the intermediates of primary carbon metabolism via phenylpropanoic acid, shikmic acid, mevalonic acid, and methyl erythritol phosphate pathways (Taiz and Zeiger 2006) ; Recently, it is reported that heat stress induces production of secondary metabolites including phenolics, flavonoids, phenyl propanoids, and plant steroids (Bharti and Khurana 1997; Wahid 2007; Wahid et al. 2008). Carotenoids show a role in protecting cellular structures in various plant species under different stress types (Havaux 1998). Studies show that lipid layer of the thylakoid membranes are stabilized and photoprotected by various carotenoids and some terpenoids such as isoprene and a-tocopherol. Exposure of plants to strong light and high temperatures caused the partitioning of xanthophylls (violaxanthin, anthraxanthin, zeax-anthins, etc.) between the light-harvesting complexes and lipid phase of thylakoid membranes and increases membrane thermostability (Havaux 1993, 1998) .

Isoprenoids are low molecular weight volatile compounds, synthesized via mevalonic acid pathway (Taiz and Zeiger 2006) ; their emission from leaves confers their role in heat tolerance (Loreto et al. 1998; Sharkey 2005). Although their synthesis is cost intensive, they show compensatory benefits in terms of heat resistance (Funk et al. 2004) ; Plants, capable of emitting higher amounts of isoprene, photosynthesize better under heat stress, which indicates a relationship between isoprene emission and heat tolerance (Velikova et al. 2004). Isoprene emission protects PSII under high temperature (Sharkey 2005), whereas the endogenous production of isoprene protects the biological membranes by directly binding with singlet oxygen (1 O2) by virtue of isoprene-conjugate double bond (Velikova et al. 2004) .

Phenolics are the largest class of secondary metabolites and include flavonoids, lignin, antho-cyanin, etc. Accumulation of soluble phenolics under heat stress is accompanied with increased activity of phenyl ammonia lyase (PAL) but decreased activity of peroxidase polyphenyl lyase (Taiz and Zeiger 2006). Acclimation to heat stress is triggered by the biosynthesis of phenolic compounds induced by high temperature (Rivero et al. 2001). They act as efficient antioxidants in plant tissues under stressful conditions (Dixon and Paiva 1995; Sgherri et al. 2004). Levels of flavonoid (e.g., anthocyanins) are greatly altered in plant tissues under heat stress (Oren-Shamir and Nissim-Levi 1999; Sachray et al. 2002; Wahid et al. 2008) .

Plant steroids, a class of secondary metabolites, also influence a variety of functions under stressful conditions. Brassinosteroids (BRs) and ginsenosides are important plant steroids whose physiological importance to high temperature tolerance in plants has been explored. Studies confirm that BRs confer tolerance to high temperature stress in brassica and tomato seedlings, by inducing the biosynthesis of major heat shock proteins (Dhaubhadel et al. 1999) . Production of ginsenosides, another important plant steroid, has been reported in all organs of Panax quinquefo-lius. It is recently reported that growing season had a great effect on the ginsenosides biosynthesis P. quinquefolius plants grown at high temperatures had 49% higher concentrations of storage root ginsenosides than respective control plants (Jochum et al. 2007; Wahid and Tariq 2008).

2.4 Molecular Responses

Transcritional regulation plays an important role in plant defense from heat stress (Singh et al. 2002). Heat stress induces numerous genes encoding transcriptional factors, which are involved in heat stress response and tolerance

(Chen and Zhu 2004; Kotak et al. 2007). Different studies revealed that several genes are up- and downregulated by abiotic stresses (Kawasaki et al. 2001; Provart et al. 2003 ; Nogueira et al. 2003). Elevated temperature affects the gene expression in storage protein synthesis and starch metabolism during grain filling stage in rice (Yamakawa et al. 2007) . Heat stress changes the pattern of gene expression, which is important for thermotolerance (Yang et al. 2006) . An account of various genes and proteins showing expression under heat stress is given below.

2.4.1 Heat Shock Genes and Proteins

Several transcriptome studies have identified many stress-responsive genes and encoding transcriptional factors during environmental stresses. Recent transgenic approaches suggest that heat tolerance is a multigenic character. Heat shock induces many genes, which are attributed to heat shock elements (HSE).These HSE are situated in the promoter region of hsp genes (Hubel and Schoffl 1994). Transgenic approach confirmed that Heat-shock transcription factor (HSF) binding to pentameric nucleotides (5'-nGAAn-3') of HSE sequences (Perisic et al. 1989; Sung et al. 2003). This HSF-HSE interaction and transcriptional activation is quite conserved in nature. This multigenic phenomenon modifying the expression pattern of transcription factors motivate a series of genes (Dong et al. 2003). Studies show that Hot1-Hot4 genes of Arabidopsis may function to improve heat tolerance; Hot1 is identified as Hsp101 in Arabidopsis thaliana (Hong and Vierling 2000). HsfA1 acts as master regulator of thermotolerance in tomato by reducing the expression of heat shock genes in co-suppression lines (Mishra et al. 2002) .

Hsfs are essential for gene expression in response to high temperature (Nover et al. 2001). Various studies show that distinctive hsp genes are not expressed in germinating pollen. Only hsp18 and hsp70 genes are transcribed in response to heat stress (Wahid et al. 2007) . A defective heat shock response of mature maize pollen was due to inefficient induction of heat shock gene transcription (Hopf et al. 1992). Enhanced expression of HSP70 assisted in translocation, proteolysis, protein translation, protein folding, aggregation, and refolding of denatured proteins (Zhang et al. 2005; Iba 2002; Weggle et al. 2004; Gorantla et al. 2007). Recent studies revealed that a-amylase genes in seeds of rice reduced the seed weight and chalkiness during ripening under heat stress (Asatsuma et al. 2006; Yamakawa et al. 2007) .

The synthesis and accumulation of heat shock proteins (HSPs) through heat shock factors (HSFs) network play great role in plant responses to heat stress (Wang et al. 2004; Kotak et al. 2007). Amounts of specific mRNA synthesis, mRNA stability, translation efficiency and alteration in protein activity increase in plants as a result of gene expression (Sullivan and Green 1993). All organisms synthesize HSPs upon exposure to high temperature. Heat stress altered gene expression in reproductive organ of plant (Dupuis and Dumas 1990; Oshino et al. 2007). Abortion of development and demarcation of pollen mother cell due to heat shock is due to tissue specific alterations in gene expression (Sakata et al. 2000; Abiko et al. 2005). In plants, a heat shock of 8-10°C above ambient temperatures induces the synthesis of both high (60-110 kDa) and low (15-30 kDa) molecular weight HSPs (Vierling 1991; Waters et al. 1996; Sun et al. 2002). These HSPs were induced either to protect the plant from injury or to help repair the injury caused by the heat stress (Leshem and Kuiper 1996) . The synthesis of HSPs occurred in different plant species when they were exposed 10-15°C above growing temperatures (Dubey 1999). Their synthesis is extremely fast, diverse, and intensive in a variety of organisms (Parsell and Lindquist 1993; Wahid et al. 2007).

Both cytosolic and organelle synthesis of HSPs has been well studied. Some HSPs that accumulate in the cytosol at 27°C and in the chloroplast at 43°C and 37°C respectively, appeared to play a role in photosynthesis and thermotolerance (Heckathorn et al. 1998b) . In maize, high temperature induced the synthesis and accumulation of chloroplast protein elongation factor EF-TU, which defended the chloro-plasts proteins from heat-induced damage (Ristic et al. 2004; Momcilovic and Ristic 2007). Maize EF-TU is a 45-46 kDa HSP confined to chloro-plast stroma is involved in development at heat tolerance in maize (Ristic and Cass 1992; Bhadula et al. 2001; Moriarty et al. 2002). In maize, heat shock of 40°C induces the synthesis of HSPs18 (Nieto-Sotelo et al. 2002). Interaction of HSPs 22 kDa with the Chenopodium album and common bean chloroplast membranes affects the composition of membrane and decreases its fluidity; thus increasing the efficiency of ATP transport (Barua et al. 2003; Simöes-Araujo et al. 2003).

Mitochondrial HSPs have been isolated from pumpkin (Cucurbita pepo) cotyledons under high temperature stress (Tsugeki et al. 1992; Kuzmin et al. 2004). They act as molecular chaperones in vitro (Schöffl et al. 1998; Guo et al. 2001; Kim and Schöffl 2002), prevent aggregation of denatured proteins (Sheffield et al. 1990), aid in folding of nascent polypeptides and refolding of denatured proteins (Lee et al. 1994; Goloubinoff et al. 1999). They also resolubilize the denatured aggregated proteins (Parsell et al. 1994). HSP68 synthesis was restricted to mitochondria as a precursor protein, but its synthesis increased during heat shock in cell (Neumann et al. 1993). When wheat, maize, and rye seedling were exposed at 42°C, five mitochondrial LMW HSPs (19, 20, 22, 23, and 28 kDa) were induced in maize and only one (20 kDa) in rye and wheat mitochondria each; the tolerance of maize was higher than wheat and rye (Korotaeva et al. 2001). The specific nucleus-encoded HSPs have been identified in potato, maize, soybean, barley, and tomato (Neumann et al. 1993; Nautiyal and Shono 2010), peas (Ko et al. 1992; Watts et al. 1992) under heat stress.

Although with less certainty, some putative functions have been assigned to HSPs when produced under normal or high temperature conditions. The rapid accumulation of HSPs may play a significant role in the safety of metabolic apparatus of the cell. Some HSPs are produced in some developing cells under control condition (Hopf et al. 1992) during embryogenesis, germination,

Fig. 6.3 Effect of heat acclimation and stress on the expression ofHSP70 in hypocotyls of different pea varieties. After Tian et al. (2009) with permission

pollen formation, fruit set and its maturation (Vierling 1991; Sun et al. 2002 Prasinos et al. 2005; Wahid et al. 2007). For instance, HSPs were produced in greater amounts in etiolated maize seedling after 5-h exposure to high temperature stress (Lund et al. 1998). Acquired ther-motolerance depends upon the synthesis of HSPs and their cellular localization (Heckathorn et al. 1999; Korotaeva et al. 2001) ; In arid and semi arid areas, plants may accumulate significant amount of HSPs in response to high leaf temperatures. In 2-day-old soybean seedlings, HSPs appeared to maintain the conformation of other proteins, as an aid for the acquired thermotoler-ance (Jinn et al. 1997) ; The wide diversity and abundance of HSPs is important for altering the plant response to high temperature stress (Waters et al. 1996). The mature pollen was susceptible to high temperature and pollen viability was extremely reduced due to nonproduction of HSPs. A distinct set of HSPs was induced in male tissues of maize under heat stress (Dupuis and Dumas 1990). HSPs (64 and 72 kDa) were induced in germinating pollens under heat stress (Frova et al. 1989). In a recent study, Tian et al. (2009) reported the improved heat tolerance of young pea seedlings due to enhanced synthesis of HSP70 (Fig. 6.3).

2.4.2 Dehydrins

Dehydrins (DHNs), belonging to subclass of LEA group II (Dure et al. 1989) , are produced at the later stages of seed development in various plant species under drought, salinity, low temperature, heat stress, nutrients deficiency, and ABA application (Close 1996; Campbell and Close 1997; Svensson et al. 2002; Wahid and Close 2007; Pulla et al. 2007; Rurek 2010). D-11 from cotton (Baker et al. 1988), RAB16 (responsive to ABA) in rice (Mundy and Chua 1988) and RAB17 in maize (Vilardell et al. 1990) were cloned and characterized as DHN genes (Campbell and Close 1997; Ismail and Hall 1999 Koag et al. 2003) . Immunological evidence indicated that DHNs are expressed in cyanobacteria (Close and Lammers 1993), brown algae (Li et al. 1997) ; liverworts (Hellwege et al. 1994), ferns (Reynolds and Bewley 1993), ginkgo (Close and Lammers 1993), and conifers (Jarvis et al. 1996). Using immunological studies, DHNs were detected in the nucleus, cytoplasm, mitochondria, chloroplasts, and vacuole (Close 1996; Campbell

SC, sclerenchyma; PH, phloem; MX, nietaxylem; PX, protosjlem; BFC, Buliform cells; LE, lower epidermis; UE, upper epidermis

Fig. 6.4 Immunohistochemical expression of dehydrins lower and upper epidermis, and buliform cells during heat in the leaf of sugarcane clone HSF-240 under control and stress, as evident from golden brown color in staining. heat stress. The dehydrins were found to associate to stele, Source: Gilani (2007)

and Close 1997; Wahid et al. 2007) and found to be associated with cytoplasmic membranes system under abiotic stresses (Koag et al. 2003). Immuno-histolocalization studies revealed that the DHNs are associated with the mesophyll, vascular, and dermal tissues of heat-stressed sugarcane (Gilani 2007, Fig. 6.4). In maize, all parts of mature embryos show dehydrin accumulation (Godoy et al. 1994). In recent studies, three low molecular weight dehydrins were reported to be expressed in sugarcane leaves in response to heat stress (Wahid and Close 2007) .

2.4.3 Senescence-Associated Genes

Temperature, pathogenic infection, drought, and nutrient deficiency; wounding and shading may increase leaf senescence (He et al. 2001). Thus about 183 senescence-associated genes (SAGs) are involved in energy metabolism, gene expression regulations, protein biosynthesis regulations, pathogenicity, stress and flower development (Liu et al. 2008) . QTLs for some senescence-related traits have been mapped on chromosome 2A, 3A, 3B, 6A, 6B, and 7A in winter wheat subjected to heat stress (Vijayalakshmi et al. 2010). A number of encoding SAGs for proteinases such as serine proteinase in parsley (Jiang et al. 1999), cysteine proteinase in Arabidopsis (Lohman et al.

1994) and aspartic proteinase in Brassica (Buchanan-Wollaston and Ainsworth 1997) are associated with leaf senescence. A large number of SAGs and defense genes has been reported to express during leaf senescence in maize (Smart et al. 1995), barley (Kleber-Janke and Krupinska 1997), rice (Lee et al. 2001), A. thaliana (Lohman et al. 1994; Oh et al. 1996; Gepstein et al. 2003), tomato (John et al. 1997; Drake et al. 1996), radish (Azumi and Watanabe 1991), and Brassica napus (Buchanan-Wollaston and Ainsworth 1997) .

Heat stress accelerates the senescence and results in decreased assimilation partitioning to grains (Spano et al. 2003). For instance, high temperature induced the expression of dehydration responsive genes (ERD1), which is known as SAG15. This gene also protects the cells from injury (Weaver et al. 1999). A combine effect of heat-shock and drought induced a senescence-associated gene (SAG12), at least in Nicotiana tabacum, which improved the stress tolerance in plants (Rizhsky et al. 2002). Heat shock (40°C) induced tmr genes in Agrobacterium, which delays the senescence. This was achieved by an inducible promoter such as HS6871 from soybean (Smart et al. 1991). Chen et al. (2002) identified 18 transcription factors such as WRKY

genes and its protein in response to senescence and environmental stresses, including heat stress, which improved the agronomic characters of crop plants.

2.4.4 Stay-Green Gene

Photosynthetic responses of annual plants can be improved by extending duration of vegetative growth and delaying leaf senescence (Thomas and Howarth 2000). Stay-green (Sgr) proteins are responsible for the green-flesh and retention of chlorophyll during senescence (Park et al. 2007; Barry et al. 2008). The trait stay-green is divided into five types such as type A, B, C, D, and E on the basis of its chlorophyll retention during leaf senescence (Thomas and Howarth 2000) . Overexpression of Sgr gene reduces the loss of chlorophyll and delays early senescence of developing leaves (Park et al. 2007). Sgr synthesis has been reported in many plants such as sorghum (Tao et al. 2000) , maize (Rajcan and Tollenaar 1999) , rice (Cha et al. 2002; Park et al. 2007), durum wheat (Spano et al. 2003), tomato (Akhtar et al. 1999; Barry et al. 2008) , pea (Sato et al. 2007) A. thaliana (Oh et al. 2000; Ren et al. 2007) , oat (Helsel and Frey 1978), and Festuca pratensis (Armstead et al. 2006).

A stay-green protein potentially downregu-lates the chlorophyll degradation at transcrip-tional level and delays senescence (Nam 1997; Park et al. 2007) . Delaying leaf senescence resulted in about 11% increase in carbon fixation in Lolium temulentum (Thomas and Howarth 2000). Tollenaar and Daynard (1978) demonstrated that some maize varieties such as L087602 shows stay-green phenotype, which increases the water, carbohydrates, and protein contents in the husks, cobs, and seeds. Nguyen (1999) demonstrated that stay-green genes delay leaf senescence in sorghum and reduce lodging in heat-stressed and low moisture areas. In fact stay-green is used as a selection criterion in warm areas (Acevedo et al. 1991; Kohli et al. 1991) , For instance, most lines of wheat are sensitive to heat stress while some lines are heat tolerant due to stay-green character (Rehman et al. 2009).

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