EXPRESSION PROFILING AND FUNCTIONAL VALIDATION OF RICE INSERTIONAL MUTANTS OsAPC6 AND Ossi

Abstract

Rice is central to the lives of billions of people around the world. Possibly the oldest domesticated grain (-10,000 years), rice is the staple food for 2.5 billion people and growing, rice is the largest single use of land for producing food, covering 9% of the earth's arable land. Rice has been a model monocot for functional genomics research due to its small genome size (430Mb) relative to other cereals, its ease of transformation, high density genetic maps, physical maps, large-scale analysis of expressed sequence tags (ESTs) and extensive synteny shared with other cereals, availability of large number of flanking sequence tags (FSTs) and economic importance. The delicately curved, long grained, highly aromatic rice which elongates and cooks soft and fluffy is categorized as Basmati which enjoys privileged treatment both in domestic and international markets coirimanding premier market price. Traditional Basmati varieties grown in India are low yielding, tall, and lodge even under low nitrogen fertilizer dose. The completion of the rice genome sequence provided a new platform for its functional genomics. Assigning functions to these genes will help greatly to improve both quality and quantity of rice. For assigning functions to the predicted genes various -approaches such as insertional mutagenesis, serial analysis of gene expression (SAGE) and RNAi have been used. A dwarf OsAPC6 with reduced seed set and insensitivity to gibberellic acid (GA) and another seedling lethal (Ossl) insertional mutant- were isolated using independent Agrobacterium mediated transformation of - Basmati 370. Thermal asymmetric interlaced PCR (TAIL-PCR) approach indicated the. position of insertion at chromosome 3 and 11 of OsAPC6 and : Ossl, respectively. Southern hybridization confirmed the single copy T-DNA- insertion in the OsAPC6 mutant. In the present study, -further characterization of these mutants was taken up using confocal laser scanning microscopy, microarray and RNAi. The role of APC6 in cell division and the reason behind reduced-seed set (45%) in the OsAPC6 mutant was elucidated using confocal laser scanning microscopic technique. Meiotic analysis at metaphase, anaphase and early telophase I of the pollen mother cells of the mutant showed normal chromosome pairing with twelve bivalents, Abstract ii normal movement of homologous chromosomes to poles .during anaphase, normal telophase and normal pollen tetrads. On an average 92-98% pollen grains were stainable and hence viable in most of the florets. The results of normal meiotic chromosome pairing and high pollen stainability similar to that of the wild type Basmati 370 indicated that microsporogenesis and male- gametophyte development were normal in OsAPC6 mutant. The embryo sac development was studied using CLSM and found that the fertilization was normal as zygote was formed and the degenerating antipodal cells were also observed. On further analysis of immature embryo sacs it was found that female gametophytes of wild type Basmati 370 had normal mitotic division while in the mutant gametophyte a cell at the micropylar end with brighter nuclei and a dividing cell at the chalazal end with two nuclei were observed. The chalazal end cell was probably .undergoing second mitosis whereas in the micropylar end cell the division - was delayed or arrested. The ' female gametophytes were arrested at three-nucleate or seven nucleate stages, indicating that loss-of-function of APC6 impairs megagametogenesis * after first or second mitotic division. There was, however, no endosperm development in a high proportion of female gametophytes of the OsAPC6.mutant due to abnormal polar nuclei formation. The reduced seed set upto 40-45% in OsAPC6 could be attributed to the absence of endosperm. Further, the RT-PCR, hpt and hygromycin resistance results showed that the mutant phenotype was due to knock-down of the. APC6 gene and mutant dwarf plants with reduced seed set could be maintained only in heterozygous condition. The expression profiling of the OsAPC6 and Ossl mutants using the microarray was studied. The effects of T-DNA insertion on the entire transcriptome of the OsAPC6 and Ossl mutants have been elucidated. The transcriptome profiles of a T-DNA insertional mutant OsAPC6 and wild type Basmati 370 was studied using microarray. The average hybridization and percentage of probe sets detected in OsAPC6 mutant was found to be 42.6% of the total 57,381 probe sets. A total of 92 genes were found to have differential expression out of.which 81 genes (88.04%) were found to be up-regulated, while only 11 genes were down-regulated. Gene Ontology studies of these genes revealed that 89% belonged - to molecular -function" (MF), 695% were of biological process (BP) and 20.6% came under the category of cellular component (CC). The expression profile of OsAPC6 mutant revealed that the genes encoding Abstract enzymes involved in GA biosynthesis were highly up-regulated. These results indicated that the biosynthesis of GA in the mutant plant is normal, or even higher than the wild type. An increase in the .chlorophyll content was further estimated in mutant as.compared to the wild type. In the microarray study a large number of genes encoding the chloroplast precursor were found to be up-regulated. The APC6 protein encoding gene (LOC_OsO3gl3370) was found to be down-regulated in the mutant. The sensing of GA is mediated by the gibberellin insensitive dwarf 1 (GID 1) receptor protein, and the complex between GA and GID 1 allows the capture of the growth-repressing DELLA protein slender rice 1 (SLR1) which forms a GA-GIDl-SLR1 complex. The SLR1 protein in this tripartite which acts as a repressor is then targeted by SCFGID2 ubiquitin complex for proteolytic degradation. This results in release of repressive state of the GA action to signal transducing state. In our microarray data, the genes encoding for GID 1 and SLR1 were found to be up-regulated, indicating that the signaling is normal till- this tripartite formation: A pathway for GA signaling where APC/C plays a role in SLR1 degradation instead of SCFGID2 complex was speculated. Since the APC6 is a part of holo-enzyme APC/C, which has a role in ubiquitin-mediated proteolytic . degradation, mutation in APC6 probably somehow does not allow the degradation of SLRI protein, thereby leading to GA-insensitive dwarf phenotype of OsAPC6 mutant. Another T-DNA insertional mutant of Basmati 370, Ossl showed 5-15% of albino seedlings during germination. These albino seedlings died soon after germination while among non-albino 10-20%died slowly without tillering after transplantation. The transcriptome expression analysis using microarray showed average hybridization and percentage of probe sets detected in Ossl mutant was found to be 42.4% and a total of 86 genes were found to have differential expression out of which 52.6% genes were found to be up-regulated. Gene Ontology studies of these genes revealed that 53.4% belong to molecular function (MF), 76.7% were of biological process (BP) and 24.4% come under the category of cellular component (CC). The expression analysis of the seedling lethal mutant revealed some genes related to the development of chlorophyll including lipoxygenase gene which is a chloroplast precursor which was found to be down-regulated by more than 10 folds. Abstract iv The domain architecture of APC6 protein using. bioinformatics studies of this protein among different organisms. ClustalW analysis and multiple alignment was done to study the conservation of this protein. In silico homology modeling of Basmati APC6 protein was done to locate the tetratrico peptide repeats' (TPR) motifs in APC6. The phylogenetic analysis and multiple alignment results of APC6 protein of Basmati 370 and other organisms revealed a high conservation of this protein from prokaryotes to eukaryotes. These results also indicated the presence. of TPR domains involved in protein-protein interaction. The TPR domains are .present in a number of proteins that are functionally unrelated, and mediate a variety of different protein-protein interactions and were discovered in the first identified APC subunits. They are also involved in chaperone, cell-cycle, transcription, and protein transport complexes. RNAi was used to silence the APC6 gene using in planta and biolistic transformation. The RNAi construct was prepared by, 'using Gateway® cloning technique in pANDA cloning vector. The target gene was PCR amplified and cloned in pENTR/D-TOPO entry vector. The ligated product'was then transformed in E. coli DH5a and the selection of recombinant colonies was done using kanamycin in growth medium as the entry vector contained. kanamycin resistance gene. The insertion of target gene was confirmed by PCR using target gene -specific primers. The plasmid DNA containing the target gene was inserted in destination vector pANDA using LR clonase from invitrogen. The ligated product was again transformed in E. coli DH5a and the selection of recombinant colony was done using kanamycin in growth medium as the destination vector has kanamycin resistance gene. The insertion was confirmed by PCR using gene specific and GUS gene primer pairs. The LR cloned product was transformed in Agrobacter'ium tumefaciens strain LBA4404 containing rifampicin resistance gene. The transformation was further confirmed using colony PCR, using gene specific primer'pairs which gave a product length of 177 bp. The construct was transformed to rice plants by two different approaches. The embryogenic rice calli were infected with pANDA vector containing the target gene by biolistics. On the other side, via in planta transformation two days old germinated rice seeds were infected with Agrobacteriuin culture containing RNAi construct. Fertile transgenic plants have been .obtained which will be used for selection of RNAi transformants using G148 antibiotic and other molecular analysis.