Investigation Of Biological Mechanisms Of Arabidopsis Root System Growth And Development In Response To L-Leu And L-Lys

Although ammonium and nitrate are the major forms of nitrogen(N)directly absorbed and utilized by plants,90%of soil N occurs in organic types,of which amino acids(AAs)being small N-containing organic compounds account for 15-60%of total soil N.The AAs can be a N nutrition taken up by plant roots and might also serve as signal molecules regulating plant growth and development.As reported by Walch-Liu et al.,L-Glu(Glu)at 50?M was enough to strongly inhibit primary root growth of Arabidopsis ecotype C24 and simultaneously stimulated the development of more lateral roots,leading to a suggestion that Glu might be a signal substance as its acting in animal system to regulate plant root growth.However,a biological mechanism(s)of AAs' effects on root system growth and architectural formation remains less reported.In our recently study on inspecting the influence of Arabidopsis root growth by 18 AAs except for Glu,certain AAs including L-leucine(L-Leu,Leu)and L-lysine(L-Lys,Lys)at lower concentrations were found to greatly reduce the primary root growth.In this work,by using combined biological approaches including root growth phenotyping on medium plates,histochemical assay,microscopic visualization of resin sections of the primary root as well as RNA sequencing-based transcriptomic analysis,we characterized in more detail root system growth-phenotype properties and subsequently identified some putatively-related molecular components for Arabidopsis root growth in response to external Leu and Lys.The major results are summarized as below:After 5 d growth of Arabidopsis seedlings(Col-0)on 1/50 B5 medium(1 mM NH4NO3 as N source)containing Leu or Lys at different concentrations,we found that 60?M Leu or Lys was enough to significantly alter root system growth phenotype with characteristics:a strong growth inhibition of the primary root(with an inhibition rate at more than 60%),increase in lateral root numbers and length,root hair-initiating position being closer to the primary root tip;the root growth of other ecotypes of Arabidopsis e.g.Ws?Ler?C24 and RLD1 showed also Leu-and Lys-sensitivity but with different degrees;the D-type of Leu and Lys and different N sources did not much affect the sensitivity of root growth to Leu and Lys.Such data suggest that Leu-and Lys-regulated root growth and architectural formation should have certain specific genetic bases.Using segment medium plates which allowed different parts of plant seedlings to contact locally with Leu or Lys,we detected that the primary root tip is the most sensitive part responsive to these two AAs.Growth recovery experiment revealed that a growth rate of the primary root,whose growth had been already inhibited by Leu and Lys,could be restored from AA-free medium to a normal level,proposing that the sensitive response of the root growth of Arabidopsis to Leu or Lys is reversible or could not be systemically memorized by plants when such AAs are absent.All together,data present here suggest that external Leu and Lys might act as a potent growth regulators or signaling molecules to modify plant root system growth and development.By means of tissue/cell anatomic analysis,histochemical staining and microscopic detection,we found that the primary root growth inhibition by Leu or Lys was mainly due to the impairment of the normality of root-tip cell division,differentiation and elongation.As compared with control,Leu-or Lys-treated plant root system showed some special anatomic natures e.g.reduction in area of primary root tip meristematic zone,elongation-and mature-zone,decline in cell size and epidermic cell numbers,lowering the meristem cell activity accompanying a reduction of auxin levels.Furthermore,we examined whether or not plant hormones,polyamines(PAs)and their biosynthetic precursor L-arginine(L-Arg)would affect the response of root growth to Leu and Lys.Resulting data showed that auxin(IAA)and jasmonic acid mehyl ester(MeJA)could greatly relief root growth from the inhibitory effect of Leu and Lys,and spermine,spermidine and Arg could even completely repress the sensitivity of the root growth to Leu and Lys,suggesting an involvement of auxin,MeJA and PAs in the process of Leu-and Lys-regulated root system development.Arg-suppressed root sensitivity to Leu and Lys might be related to the alteration/adjustment of PAs'metabolism and internal level.Moreover,we surveyed the effect of 20 glutamate receptors(GLR)on the root Leu-and Lys-sensitivity using Arabidopsis mutant lines and also the action of DNQX(glutamate receptor's antagonist)on the Leu-and Lys-response of the root growth of Arabidopsis wild type.Our results imply that a potential functional redundancy of GLRs might be one of major reasons for the observation of no effect on root Leu-and Lys-sensitivity in the individual GLR mutants,but none could exclude a possibility that GLR would participate molecular processes of Leu-and Lys-modified root system growth,since the application of DNQX did markedly reduce the Leu-and Lys-elicited root growth sensitivity.Root transcriptomic profiling using RNA-sequencing approach has respectively identified 27 and 96 differentially-expressed-genes(DEGs)in Arabidopsis seedlings treated with Leu and Lys.Among them,the genes with up-regulation seem to be involved in mainly the metabolism of hormones and AAs,the transport of sugar,phospholipids and ions like calcium,and the action of signaling-or stress-related protein kinases;the genes down-regulated are annotated to be mostly related to the metabolism of carbonhydrates,cell-wall materials,AAs and some secondary metabolites as well as signal transduction-involved kinases.Such DEGs identified here are proposed to be candidates that might involve the molecular physiology of the root system growth in response to external Leu and Lys,thus providing putative molecular targets promoting our further investigation of molecular biological processes underlying plant root growth and development.