| Plants need to sense environmental stimuli to respond adequately to changing environmental conditions,extracellular signals and invading microorganisms.During the past decades,researches have shown that plasma membrane receptors with extracellular leucine-rich repeats(eLRR)play critical roles in these procedures.Two major classes of eLRR proteins,receptor-like protein(RLP)and receptor-like kinase(RLK),form receptor complex commonly and sense CLE(CLVATA3/Embryo surrounding region-related)peptide signaling,subsequently regulate the expression of WUSCHEL-like genes,thus participate in balancing the maintenance and differentiation of shoot apical meristem(SAM),root apical meristem(RAM)and(pro)-cambium.In Arabidopsis 57 RLP genes were identified and many of them were arranged in tandem in the form of gene clusters.The fact that almost all of the RLP genes T-DNA insertion mutations were indistinguishable from wide type plants suggested that functional redundancy exist heavily among RLP genes.In this study,over-expressing RLP genes was applied to search the function of RLPs with respect to growth,development and stress response.A total of 35 over-expression constructs were generated and transformed into Col-0 and/or Ler wild type plants.42 independent transgenic T1 plants,22 independent transgenic T2 plants and 4 independent transgenic homozygote plants were collected.Our preliminary study confirmed that RLP28 was a key gene involving into the stress response,as the expression of RLP28 was elevated upon challenged with various pathogenic bacteria and diverse stress signals.Over-expressing RLP33 might enhance the biomass of plants.In addition,RLP3 and RLP11,the close homologs to RLP2 and RLP 12 respectively,functioned in balancing the proliferation and differentiation of stem cells,as over-expression of RLP3 and RLP11 can rescue the clv2 mutant.Our study implies that the strategy using over-expression is a powerful tool to dissect the RLP roles.The over-expressing transgenic lines that we obtained in this study will be of great value for further investigation of the function of RLPs.Sequencing and multiple alignments showed that 48 isolated RLP sequences were identical to the predictions in the TAIR resource,whereas 7 show alternative splicing.Furthermore,9 out of 48 showed different sequences between TAIR and NCBI resource,whereas 11 out of 48 were the same.Alternative spliced RLP genes caused shifted open reading frame(ORF),and generated new stop codons encoding truncated RLP proteins,which were supposed to abolish the RLP functions.Single nucleotide polymorphism(SNPs)among 20 ecotypes revealed that SNP prevalently distributed in RLP genes,and the number of SNPs diverse among RLP genes and ecotypes.According to the substitution characteristic,SNPs can be divided into four groups,they are 1)the Nonsyns only occur in one RLP gene in one ecotype,2)the Nonsyns lead to generate a new stop codon,3)Nonsyns and/or Syns occur in different bases encoding the AA,4)Nonsyns and/or Syns occur in the same position.Analysis on the distribution of SNPs revealed that SNPs,especially Nonsyns,mainly distributed in eLRR domains.Further analysis showed that AA substitution on the AA residue threonine(T)and L in 195 and 445 respectively in ?-strain might alter RLP2 protein structure,which was supposed to influence the function of RLP2.In Arabidopsis 32 CLE genes were identified which encoding 26 mature CLE peptides.A-type CLE peptides participate in balancing the maintenance and differentiation of SAM and RAM,while B-type CLE peptides do not.Tracheary element differentiation inhibitory factor(TDIF),which could repress xylem formation in cell culture,has the same AA sequence with CLE41 and CLE44 peptide.CLE41/44 is secreted by phloem cells and perceived by phloem intercalated with xylem/TDIF receptor(PXY/TDR),subsequently suppress the expression of WOX4(WUS-like homobox),thus promoting procambium cell proliferation and xylem formation.SdCLE is homologous to CLE41/44 and expressed in stem xylem cells.In this study,it is shown that SdCLE expressed mainly in root stele,suggesting its role in the regulation of vascular evelopment.The expression of SdCLE is also found in hypocotyl,cotyledons,trichomes and lateral root caps.In vitro assay of SdCLEp,and SdCLEG6Tp,in which the sixth glycine(G)residue was substituted with T,revealed that both could elevate the expression of ATHB8pro:GUS,which marking procambium cells and protoxylem cells,but did not disrupt its pattern,suggesting that both could promote xylem formation and procambium cell proliferation.TMO5pro:GFP marking xylem cells and its GFP density is elevated when applied with SdCLE peptide,indicating that SdCLE could promote xylem formation.Over-expressing SdCLE resulted in lignified phloem cells and increased procambium cell layers,but did not alter the vascular pattern,indicating that SdCLE could promote phloem cell lignification and procambium cell proliferation.Over-expressing SdCLEG6T leaded to lignified phloem cells and altered vascular pattern,but did not increase the procambium cell layers.The fact that SdCLEp and SdCLEG6Tp synthesized in vivo had an enhanced activity than themical stnethesis peptides could be explained by that arabinosylated form of SdCLE and SdCLEG6T might undergo post-translational arabinosylated on the seventh proline(P)residue as has been shown for CLV3.Over-expressing SdCLEG6T functioned severely than over-expressing SdCLE could be explained by that substitution G with T didn't abolish the function of SdCLE and to some extent it enhanced the ability of SdCLE... |
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