Gene Cloning And Functional Analysis Of Pectate Lyase Gene DEL1 In Rice

Pectins are the major components of the plant cell wall,which may be the most complex polysaccharide family in the living world.As the degradation enzyme of pectin,pectate lyase is responsible for the α-1,4-glycosidic linkages in demethylesterified HG by β-elimination and produces 4,5-unsaturated oligogalacturonides at their non-reducing ends.In recently,although several pectate lyase-like genes are considered to play potentially diverse physiological roles in plants,such as being expressed in anthers and pollen,and being involved in fruit softening and ripening,pathogen defense,and tissue/plant growth and development,their molecular mechanism in monocots remains largely unknown.In this study,we isolated a dwarf and early-senescence leaf 1(del1)mutant from an ethyl methanesulfonate(EMS)-mutated japonica cultivar Nipponbare.For these phenotypes,we confirmed the function of DEL1 by physiological,cytological,genetic,molecular biological and bioinformatics analyses.The main results were summarized as follow:1.del1 mutant showed growth slowly in the whole growth period and accompanied with dwarfing,shortened root,lessened tiller number and so on.Paraffin section showed that the dwarfing phenotype of del1 mutant is contributed to the reduction of cell number.Futher analysis by flow cytometry suggested that the reduction of cell number of del1 mutant depend on the delays of cell cycle progression at the G1 phase;2.del1 also exhibits a phenotype of early senescence,which becomes increasingly apparent as the plants develop.The mutant leaf apex and leaf margin exhibited a faint yellow color from 5 days after generation.With the development of plants,the young leaves were pale white and then turned green upon maturation,while the old leaves exhibited withering and cracking.TEM revealed the presence of well-developed mesophyll cells and membrane-intact chloroplasts in fully developed wild-type leaves,whereas the disordered arrangement of grana thylakoid,and the degradation of chloroplasts,was observed in del1 plants.Meanwhile,the chlorophyll content and photosynthetic rate in del1 plants was significantly lower.The expression levels of senescence-associated genes(SAGs)and related transcription factors were significantly increased.Therefore,trypan blue staining and TUNEL assay indicated that DNA degradation was widespread in the del1 mutant leaves.NBT and DAB staining indicated the accumulation of O2-and H2O2 in del1 plants,and there was also an elevated level of electrolyte leakage.Meanwhile,the activities of SOD and POD were increased in del1.The expression of ROS-scavenging genes was significantly up-regulated.All these results suggested that the accumulationof ROS caused leaf senescence in del1 mutant;3.Genetic analysis revealed that DEL1 was controlled by a single recessive gene.The F2 mapping population was generated by crossing DEL1 with Taichung Native 1,a wild-type indica variety with DNA polymorphism with japonica.Thirty F2 plants with the del1 phenotype were used for primary mapping,and DEL1 was found to be located on the long arm of chromosome 10 between markers M1 and M14.Furthermore,by using 1081 homozygous mutant plants,DEL1 was located in a 45-kb DNA region between marker M7 and M8,which include 8 ORFs.Further by sequencing analysis,one base-pair mutation was found at third exon of the ORF5.This change from G to T causes an amino acid substitution from tryptophan(Trp)to leucine(Leu).Sequence analysis indicated that DEL1 encodes a pectate lyase(PEL)precursor.The cDNA is 1476 bp in length and encodes a protein of 491 amino acid residues.Functional complementary experiment indicated that dwarf and early-senescence leaf phenotype could be rescued by the wild type allele;4.Bioinformatics analysis revealed DEL1 protein contains a PelC domain,and has stronger similarity with plant PelC.An unrooted phylogenetic tree was built among PEL members showed DEL1 are in the same clade as PMR6(susceptible to powdery mildew)of Arabidopsis.A further experiment showed that DEL1 function is not associated with plant defense.It indicated that pectate lyase-like gene has been functional divergence between monocot and dicot plants;5.DEL1 was ubiquitously expressed in all the tissues,and high levels of DEL1 expression were observed in elongation organs,such as culms,roots and sheath;6.Pectate lyase activity assay showed the activity was significantly decreased in del1 plants.Transmission electron microscopy showed that the cell wall structure of del1 mutant was significantly changed,the thickness of middle lamella and secondary cell wall was significantly decreased,while primary cell wall was significantly increased.Moreover,the pectin,cellulose,hemicellulose and seven neutral monosaccharides were significant changed.Immunohistochemistry assay showed that the degrees of methylesterification HG of del1 mutant were significantly higher than that of wild type;7.The transcriptome analysis showed that a set of the DEGs associated with cell wall and senescence was significant altered in del1 mutant indicating that DEL1 may indirectly involved in the regulation of rice development and leaf senescence associated pathway.In conclusion,our results clearly indicated that dwarfness and early-senescence leaf of del1 mutant was caused by the alternative of cell wall composition and structure.