Daye Sun

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.

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°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.

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.

The Presence of a Heterotrimeric G Protein and Its Role in Signal Transduction of Extracellular Calmodulin in Pollen Germination and Tube Growth

The role of heterotrimeric G proteins in pollen germination, tube growth, and signal transduction of extracellular calmodulin (CaM) was examined in lily pollen. Two kinds of antibodies raised against animal Gzα, one against an internal sequence and the other against its N terminus, cross-reacted with the same 41-kD protein from lily pollen plasma membrane. This 41-kD protein was also specifically ADP ribosylated by pertussis toxin. Microinjection of the membrane-impermeable G protein agonist GTP-γ-S into a pollen tube increased its growth rate, whereas microinjection of the membrane-impermeable G protein antagonist GDP-β-S and the anti-Gα antibody decreased pollen tube growth. The membrane-permeable G protein agonist cholera toxin stimulated pollen germination and tube growth. Anti-CaM antiserum inhibited pollen germination and tube growth, and this inhibitory effect was completely reversed by cholera toxin. The membrane-permeable heterotrimeric G protein antagonist pertussis toxin completely stopped pollen germination and tube growth. Purified CaM, when added directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in plasma membrane vesicles, and this increase in GTPase activity was completely inhibited by pertussis toxin and the nonhydrolyzable GTP analogs GTP-γ-S and guanylyl-5′-imidodiphosphate. The GTPase activity in plasma membrane vesicles was also stimulated by cholera toxin. These data suggest that heterotrimeric G proteins may be present in the pollen system where they may be involved in the signal transduction of extracellular CaM and in pollen germination and tube growth.

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 Receptor-Like Kinase SIT1 Mediates Salt Sensitivity by Activating MAPK3/6 and Regulating Ethylene Homeostasis in Rice

High salinity inhibits plant growth and can cause plant death. This work identified a root cell–expressed receptor-like kinase that mediates salt sensitivity by activating MAPK and upregulating ethylene synthesis, leading to an increase in the level of reactive oxygen species. High salinity causes growth inhibition and shoot bleaching in plants that do not tolerate high salt (glycophytes), including most crops. The molecules affected directly by salt and linking the extracellular stimulus to intracellular responses remain largely unknown. Here, we demonstrate that rice (Oryza sativa) Salt Intolerance 1 (SIT1), a lectin receptor-like kinase expressed mainly in root epidermal cells, mediates salt sensitivity. NaCl rapidly activates SIT1, and in the presence of salt, as SIT1 kinase activity increased, plant survival decreased. Rice MPK3 and MPK6 function as the downstream effectors of SIT1. SIT1 phosphorylates MPK3 and 6, and their activation by salt requires SIT1. SIT1 mediates ethylene production and salt-induced ethylene signaling. SIT1 promotes accumulation of reactive oxygen species (ROS), leading to growth inhibition and plant death under salt stress, which occurred in an MPK3/6- and ethylene signaling-dependent manner in Arabidopsis thaliana. Our findings demonstrate the existence of a SIT1-MPK3/6 cascade that mediates salt sensitivity by affecting ROS and ethylene homeostasis and signaling. These results provide important information for engineering salt-tolerant crops.

Crinkly4 receptor-like kinase is required to maintain the interlocking of the palea and lemma, and fertility in rice, by promoting epidermal cell differentiation.

The palea and lemma are unique organs in grass plants that form a protective barrier around the floral organs and developing kernel. The interlocking of the palea and lemma is critical for maintaining fertility and seed yield in rice; however, the molecules that control the interlocking structure remain largely unknown. Here, we showed that when OsCR4 mRNA expression was knocked down in rice by RNA interference, the palea and lemma separated at later spikelet stages and gradually turned brown after heading, resulting in the severe interruption of pistil pollination and damage to the development of embryo and endosperm, with defects in aleurone. The irregular architecture of the palea and lemma was caused by tumour-like cell growth in the outer epidermis and wart-like cell masses in the inner epidermis. These abnormal cells showed discontinuous cuticles and uneven cell walls, leading to organ self-fusion that distorted the interlocking structures. Additionally, the faster leakage of chlorophyll, reduced silica content and elevated accumulation of anthocyanin in the palea and lemma indicated a lesion in the protective barrier, which also impaired seed quality. OsCR4 is an active receptor-like kinase associated with the membrane fraction. An analysis of promoter::GUS reporter plants showed that OsCR4 is specifically expressed in the epidermal cells of paleas and lemmas. Together, these results suggest that OsCR4 plays an essential role in maintaining the interlocking of the palea and lemma by promoting epidermal cell differentiation.

A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis

Background: Na+/Ca2+ exchangers were found in animals, but no Na+/Ca2+ exchanger had been reported in plants. Results: AtNCL showed Na+/Ca2+ exchanger-like activity and regulated stress response. Conclusion: Functional Na+/Ca2+ exchanger-like protein exists in plants. Significance: Na+/Ca2+ exchange also play a role in Ca2+ homeostasis under abiotic stress in plants. Calcium ions (Ca2+) play a crucial role in many key physiological processes; thus, the maintenance of Ca2+ homeostasis is of primary importance. Na+/Ca2+ exchangers (NCXs) play an important role in Ca2+ homeostasis in animal excitable cells. Bioinformatic analysis of the Arabidopsis genome suggested the existence of a putative NCX gene, Arabidopsis NCX-like (AtNCL), encoding a protein with an NCX-like structure and different from Ca2+/H+ exchangers and Na+/H+ exchangers previously identified in plant. AtNCL was identified to localize in the Arabidopsis cell membrane fraction, have the ability of binding Ca2+, and possess NCX-like activity in a heterologous expression system of cultured mammalian CHO-K1 cells. AtNCL is broadly expressed in Arabidopsis, and abiotic stresses stimulated its transcript expression. Loss-of-function atncl mutants were less sensitive to salt stress than wild-type or AtNCL transgenic overexpression lines. In addition, the total calcium content in whole atncl mutant seedlings was higher than that in wild type by atomic absorption spectroscopy. The level of free Ca2+ in the cytosol and Ca2+ flux at the root tips of atncl mutant plants, as detected using transgenic aequorin and a scanning ion-selective electrode, required a longer recovery time following NaCl stress compared with that in wild type. All of these data suggest that AtNCL encodes a Na+/Ca2+ exchanger-like protein that participates in the maintenance of Ca2+ homeostasis in Arabidopsis. AtNCL may represent a new type of Ca2+ transporter in higher plants.

A putative leucine-rich repeat receptor kinase, OsBRR1, is involved in rice blast resistance

Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest subfamily of transmembrane receptor-like kinases in plants, and they regulate a wide variety of developmental and defense-related processes. In this study, RNA interference (RNAi) strategy was used to specifically knockdown 59 individual rice genes encoding putative LRR-RLKs, and a novel rice blast resistance-related gene (designated as OsBRR1) was identified by screening T0 RNAi population using a weakly virulent isolate of Magnaporthe oryzae, Ken 54-04. Wild-type plants (Oryza sativa L. cv. ‘Nipponbare’) showed intermediate resistance to Ken 54-04, while OsBRR1 suppression plants were susceptible to Ken 54-04. Furthermore, OsBRR1-overexpressing plants exhibited enhanced resistance to some virulent isolates (97-27-2, 99-31-1 and zhong 10-8-14). OsBRR1 expression was low in leaves and undetectable in roots under normal growth conditions, while its transcript was significantly induced in leaves infected with the blast fungus (Ken 54-04) and was moderately affected by ABA, JA and SA treatment. Overexpression or RNAi suppression of OsBRR1 did not cause visible developmental changes in rice plants. These results indicate that OsBRR1 is involved in rice resistance responses to blast fungus and mediates resistance to rice blast.