Identification Of LOBED LEAFLET1 And STAY-GREEN Genes In Medicago Truncatula

Leguminosae, the third largest family of flowing plants, grows throughout the world, and is of great economic importance for timber, fodder, drugs, and food. As for many legume species, unique advantage is their response to nitrogen limitation-nitrogen fixation from the soil by the formation of nodules, resulting in the symbiosis with rhizobia. It can reduce the fertilizer costs in agriculture. However, the genome evolution of many legume species is closely tied with history of human civilization, which made the genome duplicated and even redundant.Currently Medicago truncatula is being used as a model of legume plants since it has a modest diploid genome, self-fertile nature, relatively short generation cycle, close relationships to alfalfa and other forage legumes, and large collections of ecotypes. The genome sequencing work of Medicago truncatula has been largely developed in the past decade. A growing number of genome websites and database as well as excellent mutant populations have already been set up, which accelerates the work of M. truncatula functional genomics. As a result, the supplications of genome and transcriptome information of M. truncatula have stimulated to understand the genetics and development mechanisms in alfalfa and other legume crops.Small RNAs have been proved to play important roles in plant development. Until now, micro RNA (miRNA) and small interfering RNA (siRNA) are two kinds of small RNAs which have been well studied. microRNA is a native RNA regulator in plants. The formation of siRNAs can be from endogenous pathway and transfection of exogenous double-strand RNA. A new type of endogenous TAS3 ta-siRNA pathway was reported in Arabidopsis and rice in 2005. Exogenous siRNAs are usually used as a tool to study the target gene function, which is commonly utilized in RNAi technique.Here we have identified and characterized two kinds of developmental defect mutants-lobed leaflet1 and stay-green-from the tobacco(Nicotiana tabacum) Tnt1 retrotransposon-tagged mutant population of M. truncatula.This thesis describes the identification and molecular characterization of the genes LOL1 and SGR related with the mutant phenotypes.1. Identification of LOL1 gene and its role in M. truncatula TAS3 ta-siRNA pathway during plant developmentIn this study, we found four developmental defective mutant lines showing the same phenotypic changes. They were named loll-1 to loll-4(lobed leaflet1) respectively, because all adult leaves are lobed in these mutants. Their juvenile leaves exhibit the characteristics of adult leaves which have trifoliate form. Moreover, further analysis showed abnormal changes in flower and roots. The keel and alae split with each other and unfused central carpels resulted in the exposed ovules. The size of pollen and anther sacs was not even and some pollens were dead. During flower stage, the anthers could not open, failing in the fertilization. Observation of 12-day-old roots showed the increasing number of first and secondary order lateral roots but shorter primary roots in loll mutants compared with wild-type.LOL1 complementation and over-expression transgenic lines showed the same phenotype as that of wild-type. This result, combining with the analysis of back-crossing revealed the abnormal phenotypes was tightly linked to the LOL1 which is a single recessive locus.Phylogenetic analysis showed that LOL1 was clustered into a subgroup including the AGO7 (ZIP) in Arabidopsis and OsAGO7 (SHL4) in rice with the high similarity. In addition, LOL1 contained a central PAZ domain and a C-terminal PIWI domain which define the PPD class of proteins, also named as AGO proteins. Therefore, LOL1 is an ortholog of Arabidopsis ARGONAUTE7, which is a key gene involved in TAS3 ta-siRNA pathway.By prediction and analysis of M. truncatula TAS3 ta-siRNA pathway, we analyzed Mt miR390 cleavage manner, the TAS3 ta-siRNA genes and target genes ARFs. The result revealed that TAS3 ta-siRNA biogenesis mechanism is conserved in Medicago truncatula. These data indicate that the regulation mechanism of TAS3 tasiRNA-ARFs pathway is well conserved in both monocots and dicots and plays a fundamental role in plant development.In our study, the severe defects in both primary and lateral roots in lollmutants suggest that the root development is also sensitive to the TAS3 ta-siRNA pathway. Our data indicate that besides three ARF genes directly regulated, HD-ZIP III gene family are also regulated indirectly by TAS3 ta-siRNA pathway in root of M. truncatula. Compared with no root phenotypic changes observed in Arabidopsis ago7 mutant, defective roots of M. truncatula loll mutant showed wider range of regulation in M. truncatula development.2. Identification of M. truncatula STAY-GREEN(SGR) geneA stay-green(sgr) mutant line NF2089 was obtained by screening the tobacco Tntl-tagged mutant population of M. truncatula. The whole plants remained green during natural and dark-induced senescence. Interestingly, the stay-green phenomenon was evident not only in leaves but also in other organs, such as anther, central carpels, mature pods and seeds. After 10 days of dark-induced senescence, chlorophyll (Ch1) contents were retained much in NF2089,60% of Ch1 a and 58% of Ch1 b retained respectively. Combining with the normal decrease of the maximal fluorescence yield of photosystem II (PSII)-Fv/Fm value and retaining of thylakoid structure in senescent chloroplasts, M. truncatula sgr mutant belongs to type C stay-green mutant.SGR was cloned from M. truncatula and M. sativa SGR, the phylogenetic trees based on predicted SGR protein sequences were constructed, showing that MtSGR and MsSGR were most identical to each other, with closely similarity to PsSGR. All the SGRs from Leguminosae species including soybean, pea, M. sativa and M. truncatula were clustered together. The alignment analysis revealed that SGR family members share highly conserved central core with divergent at N and C termini. The amino acid residue Arg-145, at which the Tntl was inserted into NF2089, is an invariant residue within SGR family.MtSGR gene was constitutively expressed in all organs at low level. It is noteworthy, however, transcription was highly induced during seed maturation and nodule development with a dramatically improvement during nodule senescence. Therefore, it suggested that MtSGR is not only related with chlorophyll senescence, but may be also involved in nodule development and senescence.Altogether, we identified MtSGR gene, which is member of SGR gene family, from M. truncatula sgr mutant with stable type C stay-green phenotype during senescence. Based on the gene expression atlas, we propose that MtSGR is performed in nodule development and senescence pathway, suggesting overlap between chlorophyll senescence and nodule development.3. Down-regulation of MsSGR in alfalfa by RNA interferenceAlfalfa is regarded as important forage around the world. Hay is the most common method of forage preservation for animal feed and storage, which can cause little loss of forage material. When alfalfa in field is used as hay, the harvesting is usually fresh-cut, baled and then dried. In order to improve the quality of alfalfa, it is also important to breed stay-green cultivar whose leaf senescence can be delayed. Therefore, we cloned SGR from alfalfa (M. sativa) according to the conserved relationship between M. truncatula and alfalfa. To analyze the role of MsSGR, the MsSGR-RNAi transgenic lines of alfalfa was constructed through transformation of MsSGR-RNAi vector under the control of 35S-promoter. Five out of twelve positive transgenic lines, MsSGR-RNAi (SGRi) No.10,17,21,29, and 39 have significantly lower SGR transcription level by more than 60% compared with control line (CTRL) using qRT-PCR. The analysis of dark-induced senescence showed that the stay-green phynotype of RNAi transgenic lines also showed the characterization of type C stay-green mutant.To evaluate the potential application of MsSGR-RNAi alfalfa, we performed the mimic harvest process of these transgenic alfalfa lines. overground parts of each transgenic line and wild-type were cut and put in the field where the air is well ventilated. During the naturally dry progress, transgenic lines still stably showed the stay-green phenotype, compared with the yellowing of wild-type. Therefore, transgenic alfalfa lines have the potential predominance in harvest and post-harvest storage.We evaluated all the five MsSGR-RNAi transgenic alfalfa lines by forage analysis. It was found that No.39 has higher contents of crude protein, lower in value of ADF and NDF, compared with wild-type RSY-4D. It is commonly valued that forages with higher digestibility could supply more energy to the animal per unit of DM consumed than less digestible forages could. So the strategy of senescence delay leads to forage improvements as predicted above.In this part of study, we identified the homologs of SGR in forage alfalfa (M. sativa), and obtained stay-green transgenic alfalfa by RNA interference silencing of SGR expression. Stay-green phenotype was remained even after plants were cut off and dried. By the senescence inhibition, the forage quality analysis of transgenic alfalfa lines shows no loss but a slightly improvement, which might be considered a good sign for application of stay-green phenotype. Our work also constructs a bridge between the theoretical study of genes in M. truncatula and application of genetic improvement of alfalfa. There are few comments about alfalfa improvement of delaying leaf senescence by directly genetic manipulation. And this is the first report on applications of stay-green into legume forages.