SvCLE Polypeptide Regulates The Function Of Vascular Phloem Development In Arabidopsis Thaliana

Meristematic tissues are capable of producing new tissues and organs through continuously proliferation and differentiation.Meristematic tissues can be classified into pro-meristem,primary meristem and secondary meristem according to the developmental stages.Primary meristems are located in the tip of the roots,stems and branches,including the root apical meristem and shoot apical meristem which give rise to the underground and aerial parts of the plant,respectively.Vascular cambium,typically known as secondary meristem,forms xylem inwards and phloem outwards.CLE peptides,known as secretion peptides,are reconginzed by membrane-bound receptor(s)to modulate various plant growth and developmental processes.A total of 32 CLE genes was identified from Arabidopsis,which encoded 26 different mature CLE peptides.It has been reported that bioactive CLE peptides normally contained 12-13 amino acids which were cleavaged from the C-terminal of the CLE proteins.CLE peptides were divided into two categories,A-class and B-class CLE peptides.A-class CLE peptides are involved in regulating the balance of the proliferation and differentiation of stem cells in shoot apical meristem and/or root apical meristem,whereas B-class CLE peptides do not.In the past several years,it has been known that CLE peptides are involved in various plant growth and developmental processes.We have identified few Arabidopsis CLE genes which are highly/specifically expressed in the phloem.However,it remains unknow whether any of these CLE peptides are involved in the regulation of pholem development.To this aim,in this sudy we choosed one CLE gene,namely SvCLE,which is expressed in the pholem cell files,for further study.The function of the SvCLE peptide,its putative receptor(s)and the molecular mechanism how SvCLE mediating the phloem developement are therefore studied.The results are summaried as follows:1.SvCLE peptide could significantly inhibit the primary root length,while rp render resistence to the SvCLE peptide treatment.The results implied that RP is required for the inhibition of root length triggered by SvCLE peptide.2.SvCLE peptide could suppress APLpro:GUS signal,suggesting that SvCLE peptide inhibit the phloem cell files in roots.Furthermore,our qPCR results showed that SvCLE repressed the expression of APL in wild-type plants but not in the rp plants,implying that SvCLE-mediated the inhibition of pholem is also required for RP.3.SvCLE peptide exerted no effect on the expression of AtHB8 as showing by ATHB8pro:GUS seedlings upon the SvCLE peptide treatment.In consistence,our qPCR results indicated that the expression of AtHB8 remained unchanged upon the treatment of SvCLE peptide.Our results thus indicated that SvCLE probably exerted no effect on the development of Arabidopsis(pro-)cambium and primary xylem.4.RP is expressed in vascular tissues as showing by our pRP:GUS transgenic plants.Moreover,it is suggested by qPCRs that the APL expression is lower in rp than that in the wild-type plants.To further elucidate the role of RP in regulation of phloem development,we made rp APLpro:GUS crosses,and identified the homozygous lines subsequently.Interestingly,we found that the GUS siganl of homozygous lines were resistence to the treatment of SvCLE peptide.These results confirmed that SvCLE-triggered the abolishment of phloem is depended on RP.5.To further characterize the function of SvCLE,we isolated homozygous SvCLE T-DNA insertion lines through genotyping.However,phenotypic analyses of the homozygous mutant plants indicated that homozygous lines are indistinguishable from the wild-type plants.6.We constructed 35S:SvCLE and transformed into wild-type plants.However,we could not obtain obtaine the transgenic plants overexpressing SvCLE.Intriguingly,many healthy transgenic plants were obtained when we transformed the 35S:SvCLE construct into rp mutants.Altogether,these results further confirmed that SvCLE induced developmental defects is depended on the presence of RP.7.We could not show the interaction of BAM3 and RP by using BiFC.Therefore,it is possible that SvCLE-BAM3 and SvCLE-RP siganling pathways are independent to regulate the phloem development.In summary,our results suggested the exsitence of a SvCLE-RP signaling module,which may be independent from the SvCLE-BAM3 siganling module,to regulate the phloem development in Arabidipsis.Our results thus provided new insights on the molecular basis for the regulation of phloem development.