Thermotolerance In Arabidopsis Research Articles

Heat stress-induced decapping of WUSCHEL mRNA enhances stem cell thermotolerance in Arabidopsis

The plasticity of stem cells in response to environmental change is critical for multicellular organisms. Here, we show that MYB3R-like directly activates the key plant stem cell regulator WUSCHEL (WUS) by recruiting the methyltransferase ROOT INITIATION DEFECTIVE 2 (RID2), which functions in m7G methylation at the 5’ cap of WUS mRNA to protect it from degradation. We demonstrated that protein-folding genes are repressed by WUS to maintain precise protein synthesis in stem cells by preventing the reuse of misfolded proteins. However, upon heat stress, the MYB3R-like/RID2 module is repressed to reduce WUS transcripts via the decapping of nascent WUS mRNA. This releases the inhibition of protein folding capacity in stem cells and protects plant stem cells from heat-shock by eliminating misfolded protein aggregation. Our results reveal a tradeoff strategy in plants by reducing the accuracy of protein synthesis in exchange for the survival of stem cells at high temperatures.

Integration of C3H15-mediated transcriptional and post-transcriptional regulation confers plant thermotolerance in Arabidopsis.

Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein-RNA, protein-DNA, and protein-protein interactions. Here, we reveal an integrated multi-level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15-inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING-type E3 ligase MED25-BINDING RING-H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild-type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2-dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss-of-function and decreased in MBR2 overexpression backgrounds. Heat stress-induced accumulation of HSC70.3 promoted MBR2-mediated degradation of C3H15 protein, implying that an auto-regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops.

The temperature sensor TWA1 is required for thermotolerance in Arabidopsis

Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.

NFXL1 functions as a transcriptional activator required for thermotolerance at reproductive stage in Arabidopsis.

Plants are highly susceptible to abiotic stresses, particularly heat stress during the reproductive stage. However, the specific molecular mechanisms underlying this sensitivity remain largely unknown. In the current study, we demonstrate that the Nuclear Transcription Factor, X-box Binding Protein 1-Like 1 (NFXL1), directly regulates the expression of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A (DREB2A), which is crucial for reproductive thermotolerance in Arabidopsis. NFXL1 is upregulated by heat stress, and its mutation leads to a reduction in silique length (seed number) under heat stress conditions. RNA-Seq analysis reveals that NFXL1 has a global impact on the expression of heat stress responsive genes, including DREB2A, Heat Shock Factor A3 (HSFA3) and Heat Shock Protein 17.6 (HSP17.6) in flower buds. Interestingly, NFXL1 is enriched in the promoter region of DREB2A, but not of either HSFA3 or HSP17.6. Further experiments using electrophoretic mobility shift assay have confirmed that NFXL1 directly binds to the DNA fragment derived from the DREB2A promoter. Moreover, effector-reporter assays have shown that NFXL1 activates the DREB2A promoter. The DREB2A mutants are also heat stress sensitive at the reproductive stage, and DEREB2A is epistatic to NFXL1 in regulating thermotolerance in flower buds. It is known that HSFA3, a direct target of DREB2A, regulates the expression of heat shock proteins genes under heat stress conditions. Thus, our findings establish NFXL1 as a critical upstream regulator of DREB2A in the transcriptional cassette responsible for heat stress responses required for reproductive thermotolerance in Arabidopsis.

Arabidopsis HSFA9 Acts as a Regulator of Heat Response Gene Expression and the Acquisition of Thermotolerance and Seed Longevity.

Heat-shock transcription factors (HSFs) are crucial for regulating plant responses to heat and various stresses, as well as for maintaining normal cellular functions and plant development. HSFA9 and HSFA2 are two of the Arabidopsis class A HSFs and their expressions are dramatically induced in response to heat shock (HS) stress among all 21 Arabidopsis HSFs. However, the detailed biological roles of their cooperation have not been fully characterized. In this study, we employed an integrated approach that combined bioinformatics, molecular genetics and computational analysis to identify and validate the molecular mechanism that controls seed longevity and thermotolerance in Arabidopsis. The acquisition of tolerance to deterioration was accompanied by a significant transcriptional switch that involved the induction of primary metabolism, reactive oxygen species and unfolded protein response, as well as the regulation of genes involved in response to dehydration, heat and hypoxia. In addition, the cis-regulatory motif analysis in normal stored and controlled deterioration treatment (CDT) seeds confirmed the CDT-repressed genes with heat-shock element (HSE) in their promoters. Using a yeast two-hybrid and molecular dynamic interaction assay, it is shown that HSFA9 acted as a potential regulator that can interact with HSFA2. Moreover, the knock-out mutants of both HSFA9 and HSFA2 displayed a significant reduction in seed longevity. These novel findings link HSF transcription factors with seed deterioration tolerance and longevity.

The oxidized cellooligosaccharides confer thermotolerance in Arabidopsis by priming ethylene via heat shock factor A2.

Global climate change, especially heatwaves and aridity, is a major threat to agricultural production and food security. This requires common efforts from the scientific community to find effective solutions to better understand and protect the plant's vulnerabilities to high temperatures. The current study demonstrates the potential of cellooligosaccharides (COS), which are native and oxidized signaling molecules released by lytic polysaccharide monooxygenases (LPMO) enzymes during cell wall degradation by microbial pathogens. The extracellular perception of COS leads to the activation of damage-triggered immunity, often protecting the plant against biotic stress. However, how these signaling molecules affect abiotic stress tolerance is poorly understood. Here, we show that native COS and oxidized COS (oxiCOS) perception increase the transcript levels of several HEAT SHOCK FACTORS (HSFs) and HEAT SHOCK PROTEINS (HSPs) genes in Arabidopsis plants. However, only oxiCOS treatment triggers ethylene priming and increases thermotolerance. Furthermore, the function of the transcription factor HSFA2 is required for these processes. Altogether, our results indicate that the perception of Damage-Associated Molecular Patterns (DAMPs) may improve tolerance to adverse abiotic conditions, like exposure to high temperatures.

UBA domain protein SUF1 interacts with NatA-complex subunit NAA15 to regulate thermotolerance in Arabidopsis.

During recovery from heat stress, plants clear away the heat-stress-induced misfolded proteins through the ubiquitin-proteasome system (UPS). In the UPS, the recognition of substrate proteins by E3 ligase can be regulated by the N-terminal acetyltransferase A (NatA) complex. Here, we determined that Arabidopsis STRESS-RELATED UBIQUITIN-ASSOCIATED-DOMAIN PROTEIN FACTOR 1 (SUF1) interacts with the NatA complex core subunit NAA15 and positively regulates NAA15. The suf1 and naa15 mutants are sensitive to heat stress; the NatA substrate N SNC1 is stabilized in suf1 mutant plants during heat stress recovery. Therefore, SUF1 and its interactor NAA15 play important roles in basal thermotolerance in Arabidopsis.

PIF4 Promotes Expression of HSFA2 to Enhance Basal Thermotolerance in Arabidopsis.

Heat stress (HS) seriously restricts the growth and development of plants. When plants are exposed to extreme high temperature, the heat stress response (HSR) is activated to enable plants to survive. Sessile plants have evolved multiple strategies to sense and cope with HS. Previous studies have established that PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a key component in thermomorphogenesis; however, whether PIF4 regulates plant thermotolerance and the molecular mechanism linking this light transcriptional factor and HSR remain unclear. Here, we show that the overexpression of PIF4 indeed provides plants with a stronger basal thermotolerance and greatly improves the survival ability of Arabidopsis under severe HS. Via phylogenetic analysis, we identified two sets (six) of PIF4 homologs in wheat, and the expression patterns of the PIF4 homologs were conservatively induced by heat treatment in both wheat and Arabidopsis. Furthermore, the PIF4 protein was accumulated under heat stress and had an identical expression level. Additionally, we found that the core regulator of HSR, HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), was highly responsive to light and heat. Followed by promoter analysis and ChIP-qPCR, we further found that PIF4 can bind directly to the G-box motifs of the HSFA2 promoter. Via effector–reporter assays, we found that PIF4 binding could activate HSFA2 gene expression, thereby resulting in the activation of other HS-inducible genes, such as heat shock proteins. Finally, the overexpression of PIF4 led to a stronger basal thermotolerance under non-heat-treatment conditions, thereby resulting in an enhanced tolerance to severe heat stress. Taken together, our findings propose that PIF4 is linked to heat stress signaling by directly binding to the HSFA2 promoter and triggering the HSR at normal temperature conditions to promote the basal thermotolerance. These functions of PIF4 provide a candidate direction for breeding heat-resistant crop cultivars.

The Characteristics of Solanum lycopersicum SlSPRH1 and its Negative Role in Thermotolerance in Arabidopsis

The Characteristics of Solanum lycopersicum SlSPRH1 and its Negative Role in Thermotolerance in Arabidopsis

Hot stress: basal thermotolerance in Arabidopsis depends on two ethylene response factors, ERF95 and ERF97.

Hot stress: basal thermotolerance in Arabidopsis depends on two ethylene response factors, ERF95 and ERF97.

BRF Negatively Regulates Thermotolerance Defect of fes1a in Arabidopsis.

FES1A is a heat shock protein 70 binding protein. Mutation of FES1A leads to a defect in thermotolerance of Arabidopsis; however, independent fes1a mutants exhibit a range in the extent of thermosensitivity. Here, we found that BRF2, a gene adjacent to FES1A and encoding a component of transcription factor IIIB, affects the thermosensitivity of fes1a mutants. Knockout of BRF2 suppressed fes1a thermosensitivity, while overexpression of BRF2 increased thermosensitivity of fes1a. BRF2 in fes1a mutants regulates the transcriptional strength of RNA Polymerase II and accumulation of heat shock proteins and eventually affects the thermotolerance of fes1a. There is a cross-talking between RNA Pol III and Pol II. The cross-talking is initiated by BRF, magnified by the mutation of FES1A, and finally has an effect on thermotolerance.

AtPUB48 E3 ligase plays a crucial role in the thermotolerance of Arabidopsis

As the global temperature gradually increases, thermotolerance is vital to the growth and survival for plants. Ubiquitin-mediated protein degradation is a central regulator of many key cellular and physiological processes, including responses to biotic and abiotic stresses. E3 Ubiquitin-ligases, as the major components in the ubiquitination pathway, confer specificity of substrate recognition. Herein, we report that AtPUB48 expression was induced by heat stress, including basal and acquired thermotolerance. AtPUB48-overexpressing lines (OEs) of plants were generated to detect the functions of AtPUB48 in the heat response signaling pathway in Arabidopsis. Seeds of Atpub48-2 mutant had a lower germination rate than those of wild-type (WT) and OE plants when suffered from high temperatures. On the contrary, overexpression of AtPUB48 in Arabidopsis enhanced basal and acquired thermotolerance in seed germination and seedling growth. Moreover, the transcript expression levels of several heat-related downstream genes were highly improved in the OE lines under heat stress, although there were lower levels in the Atpub48-2 mutant compared with that of WT. An in vitro ubiquitination assay confirmed that AtPUB48 with U-box and ARM-repeats functioned as an E3 ubiquitin ligase. The subcellular localization showed that AtPUB48 localized to the nucleus. Collectively, these data imply that AtPUB48 acts as a novel regulator in the heat response signaling pathway. AtPUB48 may target the unknown substrate receptor to 26S proteasome proteolysis.

Light Primes the Thermally Induced Detoxification of Reactive Oxygen Species During Development of Thermotolerance in Arabidopsis

Reactive oxygen species (ROS) serve as critical signaling mediators in plant adaptation responses to environmental stimuli. ROS biosynthesis and metabolism should be tightly regulated, because they often impose oxidative damage on biological molecules, such as DNA and proteins, and on cellular structures. It is known that at high temperatures, ROS rapidly accumulate in plant tissues. Thus, a quick activation of ROS-scavenging systems is necessary for thermal adaptation. However, it is largely unknown how the thermo-induced ROS-detoxifying capacity is enhanced by environmental factors at the molecular level. Here, we demonstrated that environmental light primes the thermally induced ROS detoxification process for development of thermotolerance in Arabidopsis. While the ROS detoxification capacity was markedly enhanced in light-pre-treated plants at high temperatures, its enhancement was not as evident in dark-pre-treated plants. ASCORBATE PEROXIDASE 2 (APX2) is a representative ROS-scavenging enzyme that is activated under heat stress conditions. It was observed that the thermal induction of the APX2 gene was more prominent in light-pre-treated plants than in dark-pre-treated plants. Notably, the light-gated APX2 gene induction was compromised in Arabidopsis mutants lacking the red light photoreceptor phytochrome B (phyB). Furthermore, exogenous application of the antioxidant ascorbate recovered the heat-sensitive phenotype of the phyB mutant. These observations indicate that light-primed ROS-detoxifying capability is intimately linked with the induction of thermotolerance. We propose that the phyB-mediated light priming of ROS detoxification is a key component of thermotolerant adaptation in plants.

HOP family plays a major role in long‐term acquired thermotolerance in Arabidopsis

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

The role of AtPLC3 and AtPLC9 in thermotolerance in Arabidopsis

ABSTRACTPlants respond and adapt to temperature changes by many ways. In this article, we provide a supplement to previous work about AtPLC3. The subcellular localization showed that AtPLC3 was located in the plasma membrane and nucleus which differed from AtPLC9 localization. Furthermore, we measured the contents of IP3 before and after HS in AtPLC3 mutant, complemented and overexpressing lines. The results showed that the increase in IP3 after HS was partially dependent on AtPLC3 activity. To sum up, the similar expression patterns and phenotypes suggested that AtPLC3 and AtPLC9 may regulate the thermotolerance of Arabidopsis by the same mechanisms.

Histone acetyltransferase GCN5 is essential for heat stress-responsive gene activation and thermotolerance in Arabidopsis.

Exposure to temperatures exceeding the normal optimum levels, or heat stress (HS), constitutes an environmental disruption for plants, resulting in severe growth and development retardation. Here we show that loss of function of the Arabidopsis histone acetyltransferase GCN5 results in serious defects in terms of thermotolerance, and considerably impairs the transcriptional activation of HS-responsive genes. Notably, expression of several key regulators such as the HS transcription factors HSFA2 and HSFA3, Multiprotein Bridging Factor 1c (MBF1c) and UV-HYPERSENSITIVE 6 (UVH6) is down-regulated in the gcn5 mutant under HS compared with the wild-type. Chromatin immunoprecipitation (ChIP) assays indicated that GCN5 protein is enriched at the promoter regions of HSFA3 and UVH6 genes, but not in HSFA2 and MBF1c, and that GCN5 facilitates H3K9 and H3K14 acetylation, which are associated with HSFA3 and UVH6 activation under HS. Moreover, constitutive expression of UVH6 in the gcn5 mutant partially restores heat tolerance. Taken together, our data indicate that GCN5 plays a key role in the preservation of thermotolerance via versatile regulation in Arabidopsis. In addition, expression of the wheat TaGCN5 gene re-establishes heat tolerance in Arabidopsis gcn5 mutant plants, suggesting that GCN5-mediated thermotolerance may be conserved between Arabidopsis and wheat.

A Potential Role for Mitochondrial Produced Reactive Oxygen Species in Salicylic Acid-Mediated Plant Acquired Thermotolerance.

To characterize the function of salicylic acid (SA) in acquired thermotolerance, the effects of heat shock (HS) on wild-type and sid2 (for SA induction deficient 2) was investigated. After HS treatment, the survival ratio of sid2 mutant was lower than that of wild-type. However, pretreatment with hydrogen peroxide (H2O2) rescued the sid2 heat sensitivity. HsfA2 is a key component of acquired thermotolerance in Arabidopsis. The expression of HsfA2 induced by SA was highest among those of heat-inducible Hsfs (HsfA2, HsfA7a, HsfA3, HsfB1, and HsfB2) in response to HS. Furthermore, the application of AsA, an H2O2 scavenger, significantly reduced the expression level of HsfA2 induced by SA. Although SA enhanced the survival of sid2 mutant, no significant effect on the hsfA2 mutant was observed, suggesting that HsfA2 is responsible for SA-induced acquired thermotolerance as a downstream factor. Further, real-time PCR analysis revealed that after HS treatment, SA also up-regulated mRNA transcription of HS protein (Hsp) genes through AtHsfA2. Time course experiments showed an increase in the fluorescence intensity of DCF in the mitochondria occurred earlier than in other regions of the protoplasts in response to SA. The cytochrome reductase activity analysis in isolated mitochondria demonstrated that SA-induced mitochondrial ROS possibly originated from complex III in the respiration chain. Collectively, our data suggest that SA functions and acts upstream of AtHsfA2 in acquired thermotolerance, which requires a pathway with H2O2 production involved and is dependent on increased expression of Hsp genes.

Melatonin induces class A1 heat-shock factors (HSFA1s) and their possible involvement of thermotolerance in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) serves as an important signal molecule during plant developmental processes and multiple abiotic stress responses. However, the involvement of melatonin in thermotolerance and the underlying molecular mechanism in Arabidopsis were largely unknown. In this study, we found that the endogenous melatonin level in Arabidopsis leaves was significantly induced by heat stress treatment, and exogenous melatonin treatment conferred improved thermotolerance in Arabidopsis. The transcript levels of class A1 heat-shock factors (HSFA1s), which serve as the master regulators of heat stress responses, were significantly upregulated by heat stress and exogenous melatonin treatment in Arabidopsis. Notably, exogenous melatonin-enhanced thermotolerance was largely alleviated in HSFA1s quadruple knockout (QK) mutants, and HSFA1s-activated transcripts of heat-responsive genes (HSFA2, heat stress-associated 32 (HSA32), heat-shock protein 90 (HSP90), and 101 (HSP101)) might be contributed to melatonin-mediated thermotolerance. Taken together, this study provided direct link between melatonin and thermotolerance and indicated the involvement of HSFA1s-activated heat-responsive genes in melatonin-mediated thermotolerance in Arabidopsis.

Characterization of a Novel DWD protein that participates in heat stress response in Arabidopsis.

Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.

Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 3 (AtPLC3) and AtPLC9 have an Additive Effect on Thermotolerance

The heat stress response is an important adaptation, enabling plants to survive challenging environmental conditions. Our previous work demonstrated that Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 9 (AtPLC9) plays an important role in thermotolerance. During prolonged heat treatment, mutants of AtPLC3 showed decreased heat resistance. We observed no obvious phenotypic differences between plc3 mutants and wild type (WT) seedlings under normal growth conditions, but after heat shock, the plc3 seedlings displayed a decline in thermotolerance compared with WT, and also showed a 40-50% decrease in survival rate and chlorophyll contents. Expression of AtPLC3 in plc3 mutants rescued the heat-sensitive phenotype; the AtPLC3-overexpressing lines also exhibited much higher heat resistance than WT and vector-only controls. The double mutants of plc3 and plc9 displayed increased sensitivity to heat stress, compared with either single mutant. In transgenic lines containing a AtPLC3:GUS promoter fusion, GUS staining showed that AtPLC3 expresses in all tissues, except anthers and young root tips. Using the Ca(2+)-sensitive fluorescent probe Fluo-3/AM and aequorin reconstitution, we showed that plc3 mutants show a reduction in the heat-induced Ca(2+) increase. The expression of HSP genes (HSP18.2, HSP25.3, HSP70-1 and HSP83) was down-regulated in plc3 mutants and up-regulated in AtPLC3-overexpressing lines after heat shock. These results indicated that AtPLC3 also plays a role in thermotolerance in Arabidopsis, and that AtPLC3 and AtPLC9 function additionally to each other.