Rice Genetics IV - IRRI books - International Rice Research Institute

Our search for a rice homologue of PKL was also successful. This gene is expressedin panicles prior to flowering and 6 DAF, but its abundance declines sharplyat about the time when the transcripts of the LEC1-like gene appear (Fig. 7). Thisresult is compatible with the control of LEC1 by PKL in rice. It will be important todetermine whether PKL is expressed in nucellar cells at the time when we hope toinduce embryos. If it is so expressed, it may prevent ectopic LEC1 expression. We aretherefore prepared to express the antisense form of PKL to allow LEC1 expression.We shall also check whether the down-regulation of PKL in the nucellus is by itselfenough to induce transcription of endogenous LEC1.Assessing the first steps in synthetic embryo inductionIt is unclear whether ectopic expression of a single gene in the rice nucellus willinduce adventitious embryogenesis there. A more likely scenario is that several geneswill have to be expressed in a coordinated manner to achieve full embryogenesis. Weshould therefore plan to judge the effects of individual genes such as LEC1 andantisense PKL by molecular analysis rather than by microscopic examination of nucellartissue for recognizable embryos. In the case of antisense PKL, we would use insitu hybridization to assay for the accumulation of LEC1 transcripts in the nucellus.In the case of LEC1, we would use the same technique to assay for expression of thecascade of LEC1-dependent embryonic genes of rice. At present, the identities of theLEC1-dependent genes are unknown in rice but in Arabidopsis they include MADSdomainprotein AGL15 (Perry et al 1996), 12S storage proteins and ABI3 (Parcy et al1997), and six transcription factors (Kirik et al 1998, Kurup et al 2000). However, asseveral of these proteins are also FUSCA3-dependent genes, it might be necessary toexpress both LEC1 and FUS3 in the nucellus to obtain significant expression of thegenes dependent on them.Maize VIVIPAROUS1 (VP1) gene is a functional homologue of ABI3 (McCarty1995). VP1 is a transcriptional activator of the Em and C1 genes of maize. Rice genesacting downstream of LEC1 may therefore include rice homologues of VP1, Em, andC1. At least one of the abovementioned genes (encoding seed storage protein) is alsoinduced ectopically in pkl mutant and LEC1-overexpressing plants (Lotan et al 1998).It is interesting to note that naturally occurring polyembryony in the nucellus ofcitrus is accompanied by the expression of citrus seed storage proteins (Koltunow etal 1996). The marker used was a gene encoding a 33-kDa subunit of citrin, a saltsoluble,globulin fraction of Valencia seeds. The gene was expressed in polyembryonicseeds when the majority of the embryos were at the early globular stage of theembryo development. Somatic citrus embryos cultured in vivo were observed to initiate33-kDa polypeptide accumulation later in embryo development but they accumulatedthese peptides at only 10–20% of the level observed in polyembryonic seeds.Therefore, factors within the seed environment must influence the higher quantitativelevels of citrin accumulation in nucellar embryos developing in vivo, even thoughnucellar embryos, like somatic embryos, are not derived from fertilization events.394 Bennett et al