Ren-Gang Zhou

Calmodulin Is Involved in Heat Shock Signal Transduction in Wheat

The involvement of calcium and calcium-activated calmodulin (Ca2+-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°C HS. The levels of CaM mRNA and protein increased during HS at 37°C in the presence of Ca2+. The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl2 and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca2+ 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°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°C after HS at 37°C. Taken together, these results indicate that Ca2+-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.

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.

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.

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses.

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat-induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat-activated Ca²⁺-permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole-cell patch-clamp technique. The results indicated that A. thaliana cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat-shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.

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.

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.

Effects of calmodulin on DNA-binding activity of heat shock transcription factorin vitro

The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.