High temperature disturbs cellular homeostasis in plants and can lead to severe retardation of growth and development and even death. Plants exposed to heat stress exhibit a characteristic set of cellular and metabolic responses. Based on the results detailed above, the discussion focuses on the response of grape leaves to heat stress and subsequent recovery from the following aspects.
HSPs and antioxidant enzymes
One typical response to heat stress is an accelerated transcription of a set of stress or protein fate-related genes, such as those encoding HSPs, which are the main constituents of the heat shock response
. The major HSPs belong to five structurally distinct classes: Hsp100, Hsp90, Hsp70, Hsp60 and small HSPs (sHSPs)
. In this study, heat stress increased the expression of various HSPs. The transcript level of these HSPs decreased to the control level or even lower after the subsequent recovery (see Additional files
7). Under heat stress, many proteins in the cell are subject to denaturation, and HSPs function as molecular chaperones to provide protection. After the recovery, these HSPs are degraded. Moreover, a large amount of HSPs in cells resulted in the decline of transcript level of HSPs genes
. Wang et al.
 reported that the expression of protein HSP21 was induced by heat stress in grapevine leaves, and was lower than that of the control after the recovery. In plants, sHSPs form a more diverse subfamily than other HSPs/chaperones with respect to sequence similarity, cellular location and function. The sHSPs are not themselves able to refold non-native proteins, but have a high capacity to bind non-native proteins, possibly through hydrophobic interactions, and to stabilize and prevent non-native aggregation, thereby facilitating their subsequent refolding by ATP-dependent chaperones such as the DnaK system or ClpB/DnaK. Increasing evidence suggests strong correlation between sHSP accumulation and plant tolerance to stress
. Chloroplast and mitochondrial sHSPs are considered to play an important role in heat tolerance
[46, 47]. In this study, HSP22 located in mitochondria was highly expressed under heat stress and was downregulated after recovery. HSP21 located in the chloroplast was highly expressed (43-fold) by heat stress, but its transcription level declined to the control level after the following recovery (see Additional file
7). This result shows that some sHSPs may have important effects on heat tolerance of grapevines.
Among the other HSPs, over expression of HSP101 in Arabidopsis had a positive effect on growth after recovery
. It was recently found that an HSP101 homologue in Arabidopsis was involved in conferring thermotolerance to chloroplasts during heat stress
. Genetic analysis in Arabidopsis indicated that HSP101 interacts with the sHSP chaperone system to re-solubilize protein aggregates after heat stress
. Recently, it was demonstrated that the transcript level of HSP101 increased in maturing tomato pollen grains in response to heat stress
[33, 50]. In the present study, HSP101 in grape leaves was upregulated by heat stress, exhibiting a 9-fold elevated expression, but it was downregulated 6-fold after the subsequent recovery. HSP70 has essential functions in preventing aggregation and in assisting refolding of non-native proteins under both normal and stress conditions
. Some family members of HSP70 are consistently expressed and are often referred to as HSC70. These members are often involved in assisting the folding of de novo synthesized polypeptides and the import/translocation of precursor proteins. In this study, the expression of HSC70 was induced by heat stress and declined after the subsequent recovery, in agreement with previous results.
In heat stress studies, increasing attention has being paid to the generation of reactive oxygen species (ROS) and the cellular antioxidant defense systems. ROS levels are controlled by a network of enzymes and metabolites, including superoxide dismutases (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR). APX plays a pivotal role in ROS metabolism. It catalyzes the reduction of hydrogen peroxide to water by using ascorbate as a specific electron donor
. APX appears to be regulated by HSFA2
. Previous studies also indicated that it is involved in survival from high light stress
. The results of the present study showed that expression of APX, peroxidase 42 and DHAR were upregulated by 5-, 3.27- and 3.18-fold, respectively, indicating that these genes may have an important role in grape leaves in response to heat stress.
Temperature is one of the most active environmental factors affecting all plant metabolic activities, including amino acid and carbohydrate metabolism
. Secondary metabolites are involved in resistance against heat shock
. In this study, galactinol synthase transcript level increased significantly (about 50-fold) in response to heat stress and declined during recovery. Heat shock induced the production of sugars including raffinose and galactinol
. The raffinose family of oligosaccharides has been implicated in the scavenging of hydroxyl radicals
, and in protecting liposomes from desiccation through direct sugar–membrane interactions in soybean
. Galactinol synthase catalyzes the first step in the synthesis of raffinose polysaccharide, and is also regulated by HSFA2 in Arabidopsis, linking heat shock proteins and raffinose metabolism
. Weston et al.
 reported that expression of AtGolS1 was observed even at optimal temperature, and was upregulated during heat stress. Several other probe sets representing genes related to carbohydrate metabolism were regulated by heat stress in our experiment, such as invertase, UDP-glucose dehydrogenase, 6-phosphate dehydrogenase, sucrose synthase, amylase and trehalose-6-phosphate synthase/phosphatase (see Additional files
7). These enzymes are important in sugar metabolism, which were downregulated by heat stress in this study. In addition, lipid metabolism was also inhibited by heat stress, with effects on lipase, L-asparaginase, lipoxygenase and fatty acid hydroperoxide lyase.
Transcription factors (TFs)
HSFs play important roles in both basal and acquired thermotolerance through binding to cis-acting regulatory elements called heat shock element (HSEs) in the promoter region of HSP genes
. In general, plant HSFs are divided into three classes, HSFAs, HSFBs and HSFCs
. HSF30 belongs to HSFA2 which was the dominant HSF in thermotolerant cells
[39, 64] and was highly heat shock upregulated in mature tomato microspores
. The transcript levels of the HSFA2 target genes (e.g. HSP101, HSP70, HSP22, HSP17.6, and HSP15.7) were highly correlated with those of HSFA2 in Pro35S:HSFA2 Arabidopsis plants, and the induction of HSFA2 target genes was strongly reduced under heat stress in HSFA2 knockout Arabidopsis plants
. In this study, HSF30 exhibited increased expression levels (11-fold) under heat stress and was downregulated (14.3-fold) after the subsequent recovery corresponding to 16 HSPs (see Additional file
3). The present results indicated that HSF30 may also play an important regulatory role in the thermotolerance of grape. HSF7 is an HSFb2b and was strongly induced by heat stress in Arabidopsis, maize and tomato
. However, HSF7 was weakly upregulated (1.7-fold) by heat stress, but showed significant down-regulation (3.7-fold) after recovery in the present study. This indicated that HSF7 may play an important role in reducing expression of HSPs after recovery in grape leaves. In addition, HSF1 governs the expression of HSPs and regulates thermotolerance
[33, 65]. HSF1 belongs to the HSFA1a group, which was identified as a master regulator of thermotolerance in tomato
. The synthesis of members of HSP100, HSP90, HSP70, HSP60 and sHSPs under heat stress in the HSF1-RNAi strains of Chlamydomonas was dramatically reduced or completely abolished
. In the present study, HSF1 showed similar sign of change as HSF7. Therefore, HSF7 and HSF1 may play important roles in the recovery process from heat stress in grape leaves.
Qin et al.
 reported that ethylene-responsive transcriptional co-activator (ERTCA) gene is upregulated more than 8-fold in all heat treated wheat leaves. Over-expression of ERTCA enhanced heat stress tolerance of Arabidopsis
. Heat stress induced expression of ERTCA rapidly in the sensitive genotype of tomato
. In the present study, the strong induction of ERTCA expression by heat stress in grape leaves provided another piece of evidence for its role in heat tolerance. Many other transcription factor genes were also affected by heat stress and recovery in grape leaves, although their roles in heat tolerance are not clear. Interestingly, the majority of heat response transcription factors genes were responsive to the heat treatment. Most of them were downregulated. Basic leucine zipper (bZIP) transcription factors play a role in plant pathogen responses, light signaling, and ABA and abiotic stress signaling
. Our microarray data revealed that bZIP transcription factors were heat-regulated. Some were upregulated, such as Zinc finger protein-like, some were downregulated, such as B-box type Zinc finger-containing protein. GATA-type Zinc finger protein was upregulated only by the recovery treatment (see Additional file
8). Plant transcription factors WRKYs have been reported in both biotic and abiotic stress responses
. In this study, three WRKY transcription factors were downregulated by heat stress, and two WRKY transcription factors were downregulated by the recovery (see Additional files
8). These results suggested that several WRKY factors could be involved in the heat response of grape leaves. Our data indicated that expression of 3 transcription factors with NAC domain genes were also heat–regulated. Plant-specific NAC family transcription factor has a conserved NAC domain at the N-terminal of the protein and has been implicated in plant development
. It was recently reported that several NAC transcription factors were also involved in biotic and abiotic stress response
Signal transduction components
In our study, 4 genes for receptor-like kinases (RLKs) were regulated by heat stress (see Additional file
7). RLK1 is induced by wounding, pathogen attack, and salicylic acid
. Recent work indicated that RLK1 plays an important role in abscisic acid (ABA) signal transduction
. Our results suggested that heat signal transduction in grape leaves shared, at least in part, some common pathways with other biotic, abiotic and ABA stress signaling through these RLKs. Protein phosphorylation and dephosphorylation have been reported in heat signal transduction. Indeed, protein kinases and phosphatases with altered expression formed the largest group of genes. In addition, many protein phosphatases showed differential expression, suggesting that protein post-translation modification occurred during the heat response of grapevine leaves.
Calcium is a universal signaling molecule in both animals and plants, and the transient increase of Ca2+ level during heat stress is well-documented in plants
[11, 75, 76]. Heat shock triggered cytosolic Ca2+ bursts, which is transferred by Ca2+ binding proteins (CBP) such as calmodulin (CaM), CaM-related proteins, Ca2+-dependent protein kinases (CDPK), and calcineurin B-like protein (CBL), and then unregulated the expression of HSPs, due to the dependence of the final step in HSF- mediated HSP expression on a Ca2+ signal
[77, 78]. In the present analysis, candidate genes encoding the components of calcium- or calmodulin-mediated signal pathways, including calnexin, calmodulin, and CDPKs, were also heat- or recovery-regulated, suggesting a role of Ca2+-mediated signals in the heat stress response. Plant survival after severe environmental stress largely depends on the efficiency of recovery mechanisms. Among the genes activated after recovery, we found a calmodulin gene. qRT-PCR indicated a 4.28-fold induction of calmodulin transcript accumulation after the early response to salt stress