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.

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis.

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.

The Arabidopsis J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage.

AtDjB1 belongs to the J-protein family in Arabidopsis thaliana. Its biological functions in plants are largely unknown. In this study, we examined the roles of AtDjB1 in resisting heat and oxidative stresses in A. thaliana using reverse genetic analysis. AtDjB1 knockout plants (atj1-1) were more sensitive to heat stress than wildtype plants, and displayed decreased concentrations of ascorbate (ASC), and increased concentrations of hydrogen peroxide (H(2)O(2)) and oxidative products after heat shock. Application of H(2)O(2) accelerated cell death and decreased seedling viability in atj1-1. Exogenous ASC conferred much greater thermotolerance in atj1-1 than in wildtype plants, suggesting that a lower concentration of ASC in atj1-1 could be responsible for the increased concentration of H(2)O(2) and decreased thermotolerance. Furthermore, AtDjB1 was found to localize to mitochondria, directly interact with a mitochondrial heat-shock protein 70 (mtHSC70-1), and stimulate ATPase activity of mtHSC70-1. AtDjB1 knockout led to the accumulation of cellular ATP and decreased seedling respiration, indicating that AtDjB1 modulated the ASC concentration probably through affecting the function of mitochondria. Taken together, these results suggest that AtDjB1 plays a crucial role in maintaining redox homeostasis, and facilitates thermotolerance by protecting cells against heat-induced oxidative damage.

The calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana.

SUMMARY: Based on our previous findings, we proposed a pathway for the participation of Ca(2+)/calmodulin (CaM) in heat-shock (HS) signal transduction. The specific mechanism by which CaM regulates activation of heat-shock transcription factors (HSFs) is not known. CaM-binding protein kinases (CBK) are the most poorly understood of the CaM target proteins in plants. In this study, using a yeast two-hybrid assay, we found that AtCBK3 interacts with AtHSFA1a. Fluorescence resonance energy transfer was used to confirm the interaction between AtCBK3-YFP and AtHSFA1a-CFP. Furthermore, we demonstrate that purified recombinant AtCBK3 phosphorylated recombinant AtHSFA1a in vitro. We also describe the results of both downregulation of AtCBK3 expression and ectopic overexpression in Arabidopsis thaliana. The T-DNA insertion AtCBK3 knockout lines had impaired basal thermotolerance, which could be complemented by transformation of plants with the native gene. Overexpression of AtCBK3 resulted in plants with increased basal thermotolerance. Results from real-time quantitative PCR and protein gel-blot analyses suggest that AtCBK3 regulates transcription of heat-shock protein (HSP) genes and synthesis of HSPs. The binding activity of HSF to the heat-shock element (HSE), the mRNA level of HSP genes and synthesis of HSPs were upregulated in AtCBK3-overexpressing lines after HS, but downregulated in AtCBK3 null lines. These results indicate that AtCBK3 controls the binding activity of HSFs to HSEs by phosphorylation of AtHSFA1a, and is an important component of the HS signal transduction pathway.

Primary evidence for involvement of IP3 in heat-shock signal transduction in Arabidopsis.

The role of inositol 1,4,5-trisphosphate (IP(3)) in transducing heat-shock (HS) signals was examined in Arabidopsis. The whole-plant IP(3) level increased within 1 min of HS at 37 degrees C. After 3 min of HS, the IP(3) level reached a maximum 2.5 fold increase. Using the transgenic Arabidopsis plants that have AtHsp18.2 promoter-beta-glucuronidase (GUS) fusion gene, it was found that the level of GUS activity was up-regulated by the addition of caged IP(3) at both non-HS and HS temperatures and was down-regulated by the phospholipase C (PLC) inhibitors {1-[6-((17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione}(U-73122). The intracellular-free calcium ion concentration ([Ca(2+)](i)) increased during HS at 37 degrees C in suspension-cultured Arabidopsis cells expressing apoaequorin. Treatment with U-73122 prevented the increase of [Ca(2+)](i) to some extent. Above results provided primary evidence for the possible involvement of IP(3) in HS signal transduction in higher plants.

[The responses of AtJ2 and AtJ3 gene expression to environmental stresses in Arabidopsis].

By using Northern analysis of the 3''terminal noncoding region of AtJ2 and AtJ3 genes obtained by PCR, it was found that AtJ2 and AtJ3 genes were constitutively expressed in Arabidopsis roots, stems, leaves, flower buds, flowers and silique. They were expressed during the whole growth phase, but the mRNA level decreased slowly with the senescing of plants. Heat shock at 37 degrees C and cold stress at 2 degrees C induced an increase in mRNA level of AtJ2 and AtJ3, but the temporal characteristics of expression of AtJ2 and AtJ3 genes demonstrated that the response of the two genes to cold stress was much slower than that to heat shock. The expression of AtJ2 and AtJ3 genes was up-regulated slightly by water stress. Salt stress had no effect on the expression of AtJ2 and AtJ3 in the experiments. These results indicate that the expression of AtJ2 and AtJ3 genes is involved in response to many environmental stresses except salt stress.

Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis.

Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45 degrees C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.

Calmodulin-binding protein phosphatase PP7 is involved in thermotolerance in Arabidopsis.

PP7 is the first protein Ser/Thr phosphatase to be found to interact with calmodulin (CaM) in plants. The T-DNA insertion AtPP7 knockout line and AtPP7 overexpression lines were employed to study the specific function of AtPP7. The AtPP7 knockout impaired the thermotolerance of Arabidopsis seedlings while the overexpression of AtPP7 resulted in plants with increased thermotolerance. Results from real-time polymerase chain reaction (PCR) showed that the expression of AtHSP70 and AtHSP101 genes was up-regulated in AtPP7 overexpression lines after heat shock (HS) at 37 degrees C for 1 h. Protein gel blot analysis showed that HSP70 protein levels increased in AtPP7 overexpression lines after HS at 37 degrees C for 2 h. The expression of the AtPP7 gene was also induced by HS at 37 degrees C in wild-type Arabidopsis. Using a yeast two-hybrid screen, we showed an interaction between AtPP7 and CaM. In addition, we found that AtPP7 interacts with an HS transcription factor (HSF), suggesting a possible role for AtPP7 in regulating the expression of heat shock protein (HSP) genes.

Ca(2+) and calmodulin modulate DNA-binding activity of maize heat shock transcription factor in vitro.

DNA-binding activity of a maize heat shock transcription factor (HSF) was induced by heat shock of a whole cell extract at 44 degrees C. Addition of the calcium ion chelator EGTA reduced the binding of the HSF to heat shock element (HSE) in vitro. Re-addition of CaCl(2) to the sample pretreated with EGTA restored the ability of the HSF to bind to DNA. DNA-binding activity of the HSF was also induced by directly adding CaCl(2) to a whole cell extract at non-heat-shock temperature, but not by MgCl(2). During HS at 44 degrees C, calmodulin (CaM) antagonists chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) inhibited DNA-binding activity of the HSF in a concentration-dependent manner, but N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), an inactive structural analogue of W7, did not. Addition of antiserum specific to CaM reduced the binding of the HSF to HSE. Re-addition of CaM to the sample pretreated with antiserum could restore the binding activity of the HSF. DNA-binding activity of the HSF was promoted by directly adding CaM to a whole cell extract at 44 degrees C, but not by BSA. Moreover, at non-heat-shock temperature, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract, but not by BSA. Our observations further confirm the role of Ca(2+) in activation of the HSF in plant and provide the first example of the role of CaM in regulation of DNA-binding activity of the HSF. These results suggest that Ca(2+) and CaM are involved in HSP gene expression likely through regulating the activity of the HSF.

Calmodulin is involved in heat shock signal transduction in wheat.

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.