Cloning And Functional Analysis Of The Rice Lesion-mimic Associated Gene SPL29

Leaf senescence is a complex biological event that usually constitutes the last stage of leaf development, integrating multiple developmental and environmental signals. Defence responses are important for plants to fight against pathogen invasion. Many studies have been done about leaf senescence and plant defence responses, however, the molecular mechanisms involved in these two biological processes are not well understood.In this study, a new rice lesion mimic mutant spotted leaf29(spl29) was obtained, which exhibits lesion-mimic (LM) spotted leaves and rapid leaf senescence from the seedling stage throughout the rest of its life cycle. Cloning the gene responsible for spl29is hopeful to identify a novel gene involved in regulating leaf senescence and defence responses in rice, which can lay the foundation for further elucidating the molecular mechanisms of these two biological processes.Through genetic analysis, the wild-type and LM mutant phenotypes in F2populations of spl29segregated at a ratio of3:1, which suggested that the phenotype of spl29was controlled by a single recessive nuclear gene.The SPL29gene was cloned by map-based cloning. Through preliminary mapping and fine mapping, the SPL29locus was eventually limited between the markers S8and S26, a97-kb genomic region on chromosome8. Ten putative open reading frames (ORFs) were predicted according to the NCBI Blast and the annotation on RAP-DB (Rice Annotation Project Database) and RGAP (Rice Genome Annotation Project) website. By comparing genomic sequences of these ten ORFs between wild-type and spl29plants, one point mutation of G to T was identified on the eighth exon of the fifth gene, LOC_Os08g10600. This point mutation resulted in a single amino acid change from glycine (Gly) to cysteine (Cys). In order to preliminarily confirm that the G-to-T mutation was responsible for the phenotype of the spl29mutant, this nucleotide site was examined in different rice cultivars, F2non-lesion-mimic (NLM) plants and F2LM plants. The nucleotide at this site was T for the spl29and all tested F2LM plants, but G for ten different normal rice cultivars. For F2NLM plants, the nucleotide at this site was G, or both G and T. Thus, LOC_Os08g10600was a likely candidate for SPL29.A7888-bp genomic fragment of wild-type Zh11, containing the promoter, the gene region and the terminator of LOC_Os08g10600, was constructed into the functional complementary plasmid pSPL29C and transferred into the spl29calli by Agrobacterium tumefaciens-mediated transformation. The empty vector pEmvC was introduced to spl29calli as the control. After plant regeneration, all22independent transgenic lines containing the wild-type SPL29gene showed a complete rescue of the mutant phenotype, while all16independent transgenic lines with the empty vector failed to complement the spl29mutant. Therefore, functional complementation with the wild-type candidate gene rescued the mutant phenotype of spl29plants, and it was concluded that LOC_Os08g10600was the SPL29gene.Pfam analysis with its predicted amino acid sequence of SPL29showed that SPL29belongs to the UDP-glucose pyrophosphorylase gene family (PF01704). NCBI BLASTP revealed that SPL29is a putative UDP-N-acetylglucosamine pyrophosphorylase1(UAP1) gene in rice, but not the UDP-glucose pyrophosphorylase (UGP) gene. Phylogenetic analysis revealed that all UAPs in different eukaryotic organisms, including SPL29, are in a separate cluster from the UGPs. In addition, SPL29show over30%amino acid identity with all eukaryotic UAPs analysed by NCBI BLASTP. These results all suggest that SPL29encodes a putative UAP1in rice.In order to reveal whether SPL29and the mutant spl29perform UAP enzymatic activities, recombinant proteins of GST(glutathione S-transferase)-SPL29and GST-spl29were obtained with the correct molecular weights after protein expression and purification.1H-NMR (1H-nuclear magnetic resonance) spectroscopy was used to monitor the enzymatic reaction of SPL29and spl29in situ. Results showed that the reversible conversion between GlcNAc-1-P (N-acetylglucosamine-1-phosphate) and UDP-GlcNAc (UDP-N-acetylglucosamine) were observed with GST-SPL29. GST-SPL29could also catalyse the conversion of UDP-GalNAc (UDP-N-acetylgalactosamine) to GalNAc-1-P (N-acetylgalactosamine-1-phosphate). Interestingly, GST-spl29was unable to catalyse these enzymatic reactions. These NMR-based assays provide unambiguous evidence that SPL29has UAP enzymatic activities whereas the mutation of SPL29to spl29eliminates these enzymatic functions.The decrease of chlorophyll content and the depression of plant photosystem Ⅱ (PSII) function are important physiological indicators related to plant leaf senescence. Measurement results showed that chlorophyll contents in spl29leaves were decreased when compared with wild type. The senescence-induced STAY GREEN (SGR) gene plays an important role in regulating chlorophyll degradation. Analysis by qRT-PCR (quantitative real-time PCR) showed that the expression of SGR was greatly up-regulated in spl29leaves. In additon, analysis by chlorophyll a fluorescence transients revealed that the entire PSII machinery in spl29was destructed and depressed. The decreased chlorophyll content and the depressed PSII capacity indicated that the spl29plants had entered the early leaf senescence process at the physiological level.The ultrastructures of spl29mesophyll cells showed that fully developed and membrane-intact chloroplasts could be observed in many spl29mesophyll cells, while in some cells the chloroplast envelope was observed to be breaking, and mesophyll cells which had lost chloroplasts but retained mitochondria were present abundantly in spl29. These results suggest that chloroplasts developed normally in spl29at first, but were completely degraded during early leaf senescence. Chloroplast breakage may be one mechanism causing this degradation. Drastic, irreversible and complete degradation of chloroplasts in spl29leaves is coincident with the phenotype of rapid early leaf senescence to death at the cellular level.By qRT-PCR analysis, senescence-associated transcription factors, OsWRKY23, OsWRKY72and0sNAC2were up-regulated in spl29leaves. Expression levels of SAGs (senescence-associated genes), Osl2, Osl30, Osl43and Osl85, were also increased in spl29leaves. Meanwhile, nucleus encoded photosynthesis-associated genes rbcS, lhcA and lhcB, and chloroplast encoded photosynthesis-associated genes rbcL, psaA, psbA, petD, ndhA and atpA, were all down-regulated in spl29leaves. These molecular evidence of early leaf senescence in the spl29mutant suggest that early leaf senescence in this mutant is not a passive and unregulated degenerative process but an active and well-controlled genetic program.When inoculated with the bacterial blight strain PXO99, the wild-type plants showed a typical response to bacterial blight disease, while the spl29plants exhibited significantly enhanced resistance. Defence signalling-related genes, PR1a, PBZ1, PO-C1and OsWRKY45, were found up-regulated in in spl29leaves by qRT-PCR. These results suggest that plant defence responses are induced to enhancing disease resistance in the spl29mutant.Through histochemical staining, ROS (reactive oxygen species) including O2-and H2O2, were found accumulated in spl29plant leaves. Contents of malondialdehyde (MDA), an end-product of lipid peroxidation, were further measured and found increased. To explore the ROS metabolic process in spl29plants, enzymatic activities were examined for plant anti-oxidative enzymes, SOD (superoxide dismutase) and CAT (catalase). Results showed that SOD activity was increased in spl29leaves, while there was no obvious difference in CAT activity between spl29and wild-type leaves. The increase of SOD activity suggests that the spl29mutant may actively respond to the O2-accumulation and produce more H2O2, while the normal CAT activity may be not enough to scavenge the additional H2O2, leading to its accumulation in spl29. Accumulated ROS may play several important roles in the early leaf senescence and defence responses of spl29.Results also showed that two plant hormones, JA (jasmonic acid) and ABA (abscisic acid), were significantly elevated in spl29plant leaves. The accumulated JA and ABA in spl29are probably involved in its early leaf senescence and defence responses.In conclusion, functional inactivation of OsUAPl induces early leaf senescence and defence responses in spl29. ROS (O2-and H2O2) and plant hormones (JA and ABA) are all likely to be involved in these two biological processes or pathways in the mutant. It is suggested that OsUAPl may play an important role in regulating leaf senescence and defence responses in rice, and the exact molecular mechanisms need to be further studied.