Ca²⁺ Dependent Growth Mechanism And The Function Of Specific Histidien-rich Ca²⁺ Binding Protein In Leymus Chinensis

Calcium?Ca?is one of the essential elements for plant growth and development and it is also a vital signaling molecule in plants for maintaining cellular stability and sensing interior and exterior environment clues.Ecological studies found that acid rain has led to a decrease of calcium contents in the earth top soil,which threats the survival of forest plants and limnobios.The soil type of Inner Mongolia grassland is calcareous soil.From west to east,as the reduction of the organic matter content,the soluble Ca²⁺is decreasing.Sheepgrass?Leymus chinensis?Trin.?Tzvel?is a perennial forage and ecological grass,and mainly distributed in the steppes of east Eurasia and China as dominant specie.However,we know little about how the sheepgrass adapts to the physiological calcium deficient environment,and whether the sheepgrass possess specific Ca²⁺binding proteins.In this study,we developed an aseptic culture system of sheepgrass seedlings and a high-throughput Agrobacterium-mediated transient gene expression system.We studied the physiological,cellular and molecular mechanism of sheepgrass Ca²⁺nutrition,and identified physiological function of LcHRC1 in sheepgrass.The major results are followed:The sheepgrass seedlings aseptic culture system established.Sheepgrass seeds sterilized with 5%NaClO+0.1%Triton X-100 show a germination rate up to 90%.Furthermore,Agrobacteriums harboring plasmid with subcellular organelle specific RFP expression cassettes were used to transiently transform sheepgrass seedlings.Red fluorescence could be found in plasma membrane,tonoplast,mitochondria,Golgi,endoplasmic reticulum,lysosomes and plastid,which demonstrated that we have established the gene transient gene expression system in Sheepgrass.Reduction of the Ca²⁺,rather than other mineral ions,significantly promotes the elongation of the main root of sheepgrass,accompanied with H₂O₂ production and TAG accumulation in the roots.The physiological analysis showed that low concentration of H₂O₂ induced the elongation of the main root and inhibited the growth of the root hair of sheepgrass.The high concentration inhibited the growth of the main root,root hair and leaf of sheepgrass.The transcriptome sequencing found that xylosyl transferase genes related to the synthesis of primary cell wall were up-regulated in the low Ca²⁺condition.Immunofluorescence assay showed that low Ca²⁺could induce increasing of hemicellulose content in the root.Comparative transcriptomic analysis of sheepgrass found a homologous gene encoding animal histidine-rich Ca²⁺binding protein?HRC?.Bioinformatics analysis results show it was only found in the sheepgrass and sheepgrass relative wheat,and we call it LcHRC1.The LcHRC1 gene was 783 bp in length,with no intron,and encoding a protein with 261 amino acids.The 2923 bp upstream and 4056 bp downstream sequence of LcHRC1 gene were obtained by genome walking.Potential CpG islands could be identified at upstream of the transcriptional initiation site of LcHRC1,but there is no identifiable TATA box,which is coincide with its homologous gene in animal.The expression pattern of LcHRC1 in sheepgrass was also investigated and results show that it is only expressed in the root.In the animal,HRC show tissue specific expression,with high levels of expression in cardiac,skeletal,and to a lesser extend in arteriolar smooth muscles.LcHRC1 didn?t display tissue specific expression in sheepgrass.It was expresses in the leaves and roots equally.Analysis of the structure of LcHRC1 gene show that there is a histidine-rich acidic repeat region in the LcHRC1,but no cysteine-rich region was found,both of which were found in the animal HRC.Isothermal titration calorimeter?ITC200,MicroCal?analysis showed that LcHRC1 have Ca²⁺and Zn²⁺binding activities.However no Mg²⁺?Mn²⁺binding activities.By using a yeast two-hybrid system,we screened the Arabidopsis thaliana cDNA Library and found an interaction between LcHRC1 and AtTPM1 protein.AtTPM1 was predicted to be DNA-binding protein as transcription factor regulating developmental processes.The homologous protein is LcTPM1 in sheepgrass.LcHRC1 and LcTPM1 can also interact in yeast cell.We also carried out subcellular localization of LcHRC1 and LcTPM1 by fusing to the GFP protein and RFP protein,respectively.Both proteins localize in the nucleus.Arabidopsis plants expressing LcHRC1 showed significantly increasing sensitivity to exogenous ABA.However,the content of ABA showed no difference between wild type and transgenic Arabidopsis.After treated with ABA 6 hours,the level of ABI4,DREB1A and ABI5 transcripts were lower than those in the wild type.Previous researches show ABI4,DREB1A and ABI5 can serve as transcription factors regulating response to drought stress through modulating expression of corresponding genes.There are elements can be bind with DREB1A and ABI4 in LcHRC1 promoter sequerences.And the transgenic plant enhancing tolerance to drought.The ABA-induced[Ca²⁺]_cyt transient elevation in the transgenic plant was lower than that in the wild type.ABA could repress the expression of LcHRC1 in sheepgrass leaves.From all the above results we can draw a model as followed:LcHRC1 can interact with LcTPM1,and the interaction might be regulated by the binding of LcHRC1 and Ca²⁺.The interaction could regulate the level of the[Ca²⁺]_cyt,and participated in the responses to drought stress through ABA-dependent signal transduction cascades.