A2d was much higher in flower and developing seed under heat stress conditions [38]. Interestingly, in our previous study we have shown that among various class A HSFs in rice, OsHsfA2d also showed induced expression in developing rice seeds [5]. Apart from heat stress, we found that TaHsfA2d is also inducible by salt stress and by ABA [38]. Further, we demonstrate in this study that this TaHsfA2d functions as the transcriptional activator in yeast (Fig. 3). Unlike class B and class C Hsfs, class A Hsfs are known to be trans-activators due to the presence of AHA motif. We found that TaHsfA2d has a functional AHA motif and acts as transactivator similarly to other class A Hsfs.
There are 21 Hsfs in Arabidopsis, 25 in rice and 24 in Brachipodium, thus we expect 70-75 Hsfs in wheat owing to its hexaploid nature and presence of three homeologous genomes. While both Arabidopsis and tomato have a single HsfA2, rice has as many as 5 different HsfA2 (a-e) [4,12,30,45]. In both Arabidopsis and tomato, HsfA2 is highly inducible by abiotic stresses and constitutive up-regulation of this single Hsf in transgenic plants  improved the tolerance to high temperature, while its down regulation showed the negative effect [13,22,26]. Especially in Arabidopsis, overexpression of AtHsfA2 increased tolerance to several types of environmental stresses, such as high temperature [19,22,23,26], oxidative stress [16] and anoxia stress [24]. In rice also, different HsfA2 were found to be heat inducible and in addition, OsHsfA2d expressed in developing seeds [5]. Overexpression of OsHsfA2e increases the heat and salinity stress tolerance in Arabidopsis [25]. In the present investigation, Arabidopsis plants overexpressing TaHsfA2d showed remarkable tolerance to moderate high temperature provided continuously (Fig. 5 and 6). This could be due to the constitutive expression of several abiotic stresses related genes in the transgenic plants (Fig. 9). It has been shown earlier that overexpression of AtHsfA1a and A1b improved basal thermotolerance in transgenic Arabidopsis plants [46]. In the present study, a positive effect was also found on seed yield and silique length as well as silique number under moderate heat stress conditions (Fig. 6). It is known that many heat shock proteins are accumulated during seed maturation [47]. We also found higher expression of some HSPs in the siliques of transgenic Arabidopsis plants overexpressing TaHsfA2d (Fig. 9) such as HSP 26.5P, HSP 101, HSP70-T2, HSP 23.6-M and HSP17. Kotak et al. [48] found that accumulation of HSPs in developing seeds is regulated by AtHsfA9 in Arabidopsis. Whether TaHsfA2d also governs accumulation of HSPs in wheat developing grains remains to be elucidated.
Overexpression of TaHsfA2d in Arabidopsis also increased plant tolerance against salinity and drought stress, as reflected by better germination, higher chlorophyll retention and biomass accumulation ( Fig. 7 and 8). This can again be explained by the constitutive expression of several salt/osmotic stresses related genes in transgenic plants (Fig. 8). Sun et al. [49] reported that overexpression of molecular chaperon HSP17.7CII increased salt and osmotic stress tolerance. Overexpression of GolS1 increased water stress tolerance in Arabidopsis through accumulation of higher amounts of raffinose [50]. In rice, it has been shown that myo-inositol 1-P-synthase (MIPS) gene has important protective functions under salinity stress [51,52]. We have recently found that in heat stress also, the levels of myo-inositol increase significantly and which is also correlated well with TaMIPS expression in wheat [53]. It is therefore likely that salinity stress tolerance showed by Arabidopsis plants overexpressing TaHsfA2d is due to higher expression of these genes (Fig. 9).
Target genes for HsfA2 were identified through microarray analysis in transgenic plants overexpressing Arabidopsis HsfA2 [16] and rice HsfA2e [25]. It has been found that Arabidopsis HsfA2 governs expression of 46 genes, which were constitutively expressed in transgenic plants. Similarly, 37 genes were constitutively up-regulated in transgenic Arabidopsis plants overexpressing rice OsHsfA2e. There is a fair similarity in the diversity of genes governed by these two Hsf A2. On the basis of these reports we checked the expression of 21 target genes of HsfA2. These genes mainly represent heat shock proteins, reactive oxygen scavengers and other stress related proteins. We found that 16 out of 21 genes examined, have constitutively higher expression in seedling, flower and siliques of transgenic Arabidopsis plants overexpressing TaHsfA2d. A chloroplastic sHSP gene showed remarkably high expression in all the tissues tested (Fig. 9). Recently, we have shown that over-expression of a wheat chloroplastic sHSP26 improves photosynthetic efficiencies under heat stress conditions in transgenic Arabidopsis plants and also take part in seed development [54]. It is worth mentioning here that in our previous report [38], we reported many seed specific genes induced by heat stress, are also constitutively expressed in transgenic Arabidopsis plants overexpressing TaHsfA2d, such as sHSPs, HSP70, HSP101 and Sti. It is therefore likely that heat, drought and salt stress tolerance of the transgenic Arabidopsis plants overexpressing TaHsfA2d is provided by constitutive expression of these stress related genes.
Arabidopsis plants overexpressing TaHsfA2d, which preferentially expresses in developing wheat grains, showed improved heat and salt stress tolerance at various growth stages, and higher yields under moderate heat stress conditions. These findings suggest that TaHsfA2d gene may be useful in molecular breeding of wheat for enhanced thermotolerance especially during terminal heat stress. Figure S1 Cloning of wheat Hsf cDNA and Genomic clones. 5 and 3 RACE-PCR was done by using RNA from developing seeds from the heat stressed plants. Both 5and 3RACE-PCR was performed using SMART TM RACE amplification kit (Clontech, Palo Alto, USA) as per the manufacturer's instructions. The resultant PCR fragments were then cloned using pGEM-T Easy vector (Promega, USA) and sequenced. For cloning of genomic fragment, primers were designed from cloned full-length cDNA and PCR was conducted by using wheat genomic DNA as template by using Long-PCR Enzyme mix (Fermentas, Lithuania). The PCR products were purified using the PCR purification kit (Qiagen, Germany) and cloned into the pGEM-T Easy vector and then sequenced.Lemon Blueberry Pancakes
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