Calcium Signal And Heat Shock Protein Mediate Tolerance In BADH Transgenic Plainllltss

Glycine betaine (GB) is a substance with special efficiency of the compatibility. GBincrease plant tolerance at the same time accompanied by heat shock protein (HSP, heat shockprotein) accumulation in plants under abiotic stress. HSPs can act as chaperones of denaturedproteins and assist in the translocation and/or degradation of damaged proteins under variousstresses. GB improves salt resistance related to signal transduction and ion homeostasis.Based on the previous work, this study used these two kinds of genetically modified materialand processing method of exogenous betaine to analysis the role of GB and different adversitystress and the relationship between the different genetic background, to explore the transgenicplants for heat resistance and heat shock protein and calcium and calmodulin signalingpathways, the relationship between GB how to protect the photosynthetic mechanism andinfluence the expression of HSP and improve resistance and active oxygen removal system.(1) To investigate the mechanism of how GB influences the expression of HSP, both theaccumulation of GB in vivo and exogenously applied GB in WT seedlings was studied duringNaCl stress. The elongation zone in tobacco root epidermal cells treated with NaCl. Atransient Ca2+efflux was found after NaCl treatment for1-2min in the epidermal cells of theelongation zone of tobacco roots. But increasing the NaCl concentration (50-100mM) did notsignificantly increase the speed of the outflow of calcium ions. In addition, Ca2+-ATPasemetabolic inhibitors (Eosin Y; eryth-B and CPA) had not significantly effect on Ca2+efflux,which indicated the transient NaCl-induced Ca2+efflux were likely produced by the cell wallcation exchange. After24h of NaCl treatment, an influx of Ca2+was observed, meanwhile lowconcentrations of GB significantly increased NaCl-induced Ca2+influx. Pharmacologyexperiments showed that GB can enhance Ca2+influx through the LaCl3sensitive calcium ionchannel under stress condition.(2) GB increased intracellular free calcium ion concentration ([Ca2+]cyt) using LSCM,which proved whether foreign or internal composition GB can enhance [Ca2+]cyt.The resultsof non-invasive microelectrode ion flux measuring technique and LSCM proved there is the causal relationships between GB and calcium from two aspects of the static and dynamicunder salt stress and high temperature stress condition.(3) GB increased the calcium content of tobacco plants under salt or heat stress. Tofurther investigate whether GB plays a role in Ca2+uptake, the calcium content of WT plantspre-treated with GB (WT+GB) and transgenic plants (T) were compared with that of WTplants. GB affected Ca2+acquisition in shoots and roots during long-time NaCl stress, and thecalcium content of WT plants pretreated with GB and that of T plants was higher than that ofuntreated WT plants. The results were consistent with the calcium content of leaves underheat stress.(4) GB increased the intracellular free calcium ion concentration and enhanced theexpression of calmodulin (CaM) and heat shock factor (HSF) genes resulting in potentiatedlevels of heat shock proteins (HSPs). Pharmacological experiments confirmed that Ca2+andCaM increased the HSFs and HSPs gene expressions coincide with increased the levels ofHSP70accumulation. The causality of GB, calcium-calmodulin (Ca2+-CaM) and HSP isconfirmed from gene expression level by real-time quantitative PCR. It is proved that GB canenhance the levels of HSP70accumulation through the Ca2+-CaM signal pathway from thelevel of protein by Western blotting. A possible regulatory model of Ca2+-CaM in the signaltransduction pathway for induction of transcription and translation of the active HSPs is described.(5) Significant accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2-)were observed in wild type plants under heat stress; however, these accumulations were muchless in transgenic plants. An important finding reported herein is that exogenous GB cannotdirectly reduce the content of ROS. However, enzyme extraction from the WT can slightlyreduce ROS. In particular, enzyme extraction from transgenic plants greatly decreased theROS compared to the WT plants. These results also indicate that GB indirectly scavengesROS through antioxidative defense in vivo.(6) GB increase ROS scavenging enzyme activities. Evidently, GB may enhance theactivities of these enzymes to quench ROS in vivo, resulting in a lower ROS concentration.The activities of antioxidant enzymes were also stronger in accordance with lower relativeelectrolyte conductivity (REC) and malondialdehyde (MDA) content in transgenic lines,indicating that the degree of membrane injury in transgenic plants was lower than that in wild type plants. In addition, GB enhanced the expression of antioxidant enzyme genes. The resultssuggested that the accumulation of GB in vivo cannot directly eliminate reactive oxygenspecies (ROS), rather, through maintaining higher activities of antioxidant enzymes to lessenthe accumulation of ROS in transgenic plants and decrease the degree of membrane injury.(7) GB increase ROS scavenging enzyme (CuSOD, APX1and CAT1) gene expression.Pharmacological experiments confirmed that Ca2+and CaM increased these genes expressions.It is confirmed that GB can affect a series of gene expression through Ca2+-CaM pathway byreal-time quantitative PCR.(8) The chlorophyll fluorescence analysis of wild type and transgenic plants exposed toheat treatment (42°C) showed that transgenic plants exhibited higher photosynthetic capacitythan wild type plants. This result suggests that the accumulation of GB increased the toleranceto heat-enhanced photoinhibition. This increased tolerance is associated with theimprovement of D1protein content, which accelerated the repair of photosystem II (PSII)from heat-enhanced photoinhibition. GB also increased the tolerance to NaCl-enhancedphotoinhibition.GB is researched in different plant genetic background for salt stress resistance and hightemperature mechanism, to confirm biological function of GB. The relation of GB, HSP andCa2+-CaM signal transduction pathways is clarified from the heat shock protein accumulation,signal transduction, active oxygen removal, photosynthetic mechanism as well as theprotection of related gene expression. Environmental stresses generate ROS accumulation toformate denatured protein to affect normal metabolic activity. These results suggests thatstress signals are perceived by an unidentified receptor and GB applied exogenously oraccumulated in vivo in BADH-transgenic plants may act as a cofactor to activate Ca2+channels in the plasma membrane or intracellular Ca2+store membrane resulting in anincrease in [Ca2+]cyt. GB accumulation can strengthen this signal, This moderately elevatedlevel of [Ca2+]cytpromoted the expression of CaM1, which increased the DNA-bindingactivity of HSF. Activation of HSF initiated transcription and translation of HSP genes. Theelevated HSP, as molecular chaperone, assist in modified protein renaturation, reducedenatured protein accumulation, to improve plant resistance. In addition, GB induced theexpression of ROS scavenging enzyme, also reduce the accumulation of ROS to alleviate the damage caused by stress. Other pathways are possible including the regulation of HSFphosphorylation by regulation of CaM-dependent kinase, CDPK, MAPK activity, etc.; futurestudies may determine definitively which pathways are playing a role.