Progress toward the functional analysis of TGS1 genes in Arabidopsis thaliana

ORTIZ JPA; SIENA LA; SELLES B; MICHAUD C; PESSINO SC; INGOUFF M; LEBLANC O

Bahía Blanca

Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2016

CERZOS-CONICET

Apomixis refers to various reproductive behaviors of angiosperms that result in asexual reproduction through seeds. Apomictic reproduction avoids both meiotic reduction and egg cell fertilization, generating offspring that are exact genetic replicas of the mother plant. The transfer of apomixis to cereals and others crops would greatly benefit agriculture by allowing the fixation of hybrid vigor and clonal multiplication by seeds of the best hybrids. However, to achieve this, a better understanding of both the cellular and molecular bases underlying apomictic development is required. Recently, our research group reported evidence for the association between the deregulation of several genes encoding RNA methyltransferases and apomictic development in tetraploid Paspalum notatum genotypes. One of these genes corresponds to a TGS1 (trimethyl guanosylsynthase-1) homolog, named TGS1-like, which expression pattern is positively correlated with the rate of sexuality. While TGS1 is conserved across all eukaryotes and contains a single methyltransferase domain, TGS1-like is a plant-specific gene, which includes an additional WW domain. The function of both TGS1 and TGS1-like remains uncharacterized in plants, but TGS1 performs a dual activity in yeast, animals and insects: 1) it trimethylates the 5' end of specific sn(o)RNAs involved in splicing, rRNA processing and telomerase function; and 2) it acts as a co-activator associated with PRIP in several transcription mediator complexes. In yeast, TGS1 is necessary for cell cycle progression, so defective mutants show delayed growth at low temperatures. In animals, the lack of TGS1 was associated with defective meiosis and an impaired hepatic function. The objective of this work was to functionally characterize two tgs1-like mutants in Arabidopsis. RT-PCR revealed preferential expression in reproductive tissues at pre-sporogenesis, sporogenesis and gametogenesis. Selfing of heterozygous lines and progeny genotyping revealed a strong segregation bias against both mutant and heterozygous classes (1hm:2ht:2wt), indicative of defective transmission of mutant alleles. Siliques of heterozygous and homozygous plants showed in average 51% and 62% of aborted seeds, respectively, suggesting gametophytic effects. Reciprocal crosses between homozygous and wild type plants revealed 37% of aborted seeds when the mutant allele is transmited through female gametes and 18% when transmitted through male gametes, suggesting that the female gametophyte was preferentially affected. In order to characterize TGS1-like expression patterns in Arabidopsis wild type plants were transformed with synthetic constructs carrying pTGS1-like/TGS1-like/GUS or GFP. TGS1-like is expressed in sub-terminal root tips, lateral root meristem, stomata, ovule primordia, undifferentiated gametophytes, egg cells and immature embryos. Cytoembryological analysis revealed developmental defects during megagametogenesis, endosperm and embryo development. Symmetry alterations were detected on the suspensor and the proper embryo. A delay in endosperm development was also evident. Finally, we are exploring TGS1-like function using different biochemical approaches including: complementation tests in yeast defective for TGS1 using different combinations of TGS1-like domains; immunoprecipitation of TGS1-like protein and RNA partner in pTGS1-like/TGS1-like/GUS transformants; and immunolocalization in reproductive tissues using a monoclonal antibody. The knowledge gained from this work will contribute to our understanding of the role of TGS1 proteins during reproduction in plants and to the harnessing of apomixis in crops.