| There are two research subjects in this paper. One is functional analysis of a key tillering regulatorTE and another is map-based cloning of LMS1in rice. Tillering, the formation of lateral shoots, is animportant determinant of rice architecture and crop yield, and is a complex process that is regulated byseveral genetic pathways. There we identified a key rice tillering regulator tiller enhancer (TE).Through functional analysis of TE our findings uncovered a new molecular mechanism of rice shootbranching. Partial cell necrosis in lesion mimic mutants is a type of programmed cell death,there weisolated a rice lesion mimic mutant lms1(lesion mimic and senilism1) with spreading cell necrosisthrough all growth stages. Using map-based cloning we cloned the LMS1gene encoding hydroperoxidelyase (OsHPL3) which is an essential enzyme involving in plant LOX-HPL pathway. The loss-of-function of LMS1leads to the lesion mimic phenotype. Results from our experiments aresummarized as follows:1. Compared to WT, te mutant had much more tillers and the capability of producing newtillers is sustained from tillering stage to pustulation period in te mutant. Besides, te mutant alsodisplayed much shorter internodes, twisted flag leaves and panicles, and low seed-setting rate.Histological observation showed that the dwarf stem and smallish leaves in te mutant were notcaused by cell size but the reduction of cell number.2. The te locus was previously mapped to the rice chromosome3within23kb region. Twomutants, a deletion of nucleotide38-C and a substitution of nucleotide40-C to G downstream ofthe ATG initiation codon, were identified in the first exon of predicted gene LOC-Os03g03150.Genetic complementation completely rescued the abnormal phenotype of te mutant. Thus weconcluded that LOC-Os03g03150corresponds to TE gene. mRNA in situ hybridization andGUS-staining analysis demonstrated that TE is primarily expressed in the shoot apical meristem,axillary meristem, root meristem, lateral buds, young leaves and vascular tissue. Transientexpression in Arabidopsis protoplasts revealed that the TE-GFP fusion protein is mostly targetedto the nucleus.3. Phylogenetic analyses revealed that TE encodes a rice homologue of Cdh1class proteins,which are co-activators of the anaphase promoting complex/cyclosome (APC/C) E3ubiquitinligase, and the primary function is to promote mitotic exit and maintain in G0/G1stage.Overexpression of TE in Schizosaccahromces prombe inhibited cell division and caused cellelongation. Consistent with this observation, flow cytometry and DAPI staining analyses showedthat some cells in the te flag leaves had a4C DNA content and were binucleated compared to WT.These results support the notion that TE encodes a Cdh1class protein and it plays anevolutionarily conserved role in cell cycle regulation.4. It's previously reported that MOC1is a key factor promoting tillering in rice and we founda typical D-box domain at its N-terminal, which is recognized by Cdh1for ubiquitination. Biomolecular fluorescence complementation (BiFC) assay showed that TE interacted with MOC1in Nicotiana benthamiana leaf cells. Moreover, the transgenic plants overexpressing of TE causeddecreased tiller numbers with reduction of MOC1accumulation. Taken together, these resultssupport the hypothesis that TE is a negative factor of rice tillering, it probably mediated theubiquitination and subsequent degradation of MOC1protein by26S proteasome to controltillering in rice.5. Rice lesion mimic mutant lms1developed white spotted lesion mimicry at3thto4thleafstage, and then the lesion spread all of the leaves and results in the senescnece of rice leaves.DAB and trypan blue staining showed that the cell necrosis in lms1leaves was a type of PCDwith reactive oxygen species accumulations. Aniline blue staining displayed that a lot of callosewas deposited in the mesophyll cells. Besides, the shading experiment showed that the lesion mimicphenotype of lms1mutant was caused by light intensity.6. Genetic analysis revealed that the lms1mutant is controlled by a single recessive gene.Then we fine-mapped LMS1gene to55kb region on short arm of chromosome2with2912"mutant×9311" F2and F3recessive individuals, including8open reading frames (ORFs).Sequence analysis showed that gene ORF7(LOC_Os02g02000) had an insertion of1896bpexogenous DNA fragment after the509nucleotide away from the ATG, which resulted in the lossof function of gene LMS1. Genomic complementary test confirmed that the abnormality of geneLMS1resulted in the lesion mimic phenotype in lms1mutant plants.7. Protein sequence blast showed that LMS1encodes the hydroperoxide lyase (OsHPL)which is an essential enzyme involved in plant lipoxygenase (LOX) pathway. It cleaved13-HPOT to generate volatile (Z)-3-hexenal and its derivatives and nonvolatile C12-oxo fatty acid.GC-MS showed that there is almost no C6volatile substance ((E)-2-hexenal,(Z)-3-hexenol) in theleaves of lms1mutant, which indicated that OsHPL3is a key HPL member in the LOX-HPLpathway in rice leaves.8. Compare to WT, the content of lipid hydroperoxides (LHP) in the flag leaves of lms1mutant was much higher at the flowering stage with nearly5.3-fold than that of WT.We supposedthe loss-of-function of LMS1in the lms1mutant resulted in the lack of essential13-HPL in theleaf cells to catalyse the subsequent cleavage reaction of13-hydroperoxy fatty acid. As a result, alot of LHP was deposited in the leaf cells in lms1mutant and caused the initiation of lesion mimicphenotype.
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