In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of ~1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(ma)17-prp(ma}27 with four phenotypic classes of mutants and 21 mutants in the prp2-prpll complementation groups (formerly ma2-mall]. The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(ma}2-prp(ma)ll mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate oi splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(ma)2-prp(ma)ll groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing. The precise removal of the intron from nuclear premRNA is an essential process in eukaryotic gene expres sion. The splicing reaction takes place in a complex par ticle termed the spliceosome (Brody and Abelson 1985;Frendewey and Keller 1985;Grabowski et al. 1985). The fimction of the spliceosome in nuclear pre-mRNA splicing is to align the splice sites and to catalyze the two-step splicing reaction. Therefore, to understand mRNA splicing, it will not only be necessary to eluci date the components and their functions but also the pathway of assembly of the spliceosome.The process of pre-mRNA splicing appears to be very similar in the yeast and the mammalian systems, but a particular advantage to the study of splicing in yeast is the facility with which a genetic approach can be applied to this problem. Not only can mutants defective in splicing be used to enumerate the components and their interactions but they can be useful in delineating steps in the assembly of the spliceosome. Before the process of pre-mRNA splicing was discovered, a set of tempera ture-sensitive mutants in RNA synthesis was found that defined the RNA2-RNA11 genes (Hartwell 1967). Sur prisingly, all of these mutants are defective in mRNA splicing (for review, see Warner 1987; Vija3nraghavan and Abelson 1989). By general consensus among the commu nity of researchers working on RNA processing in yeast and the yeast genetic stock center, these mutants will henceforth be called pre-RNA processing [prp] mutants.

SummaryGrass flowers are highly derived compared to their eudicot counterparts. To delineate OsMADS1 functions in rice floret organ development we have examined its evolution and the consequences of its knockdown or overexpression. Molecular phylogeny suggests the co-evolution of OsMADS1 with grass family diversification. OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea, creating glume-like features, with severe derangements in lemma differentiation. Conversely, ectopic OsMADS1 expression suffices to direct lemma-like differentiation in the glume. Strikingly, in many OsMADS1 knockdown florets glume-like organs occupy all the inner whorls. Such effects in the second and third whorl are unexplained, as wild-type florets do not express OsMADS1 in these primordia and because transcripts for rice B and C organ-identity genes are unaffected by OsMADS1 knockdown. Through a screen for OsMADS1 targets we identify a flower-specific Nt-gh3 type gene, OsMGH3, as a downstream gene. The delayed transcription activation of OsMGH3 by dexamethasone-inducible OsMADS1 suggests indirect activation. The OsMGH3 floret expression profile suggests a novel role for OsMADS1 as an early-acting regulator of second and third whorl organ fate. We thus demonstrate the differential contribution of OsMADS1 for lemma versus palea development and provide evidence for its regulatory function in patterning inner whorl organs.

Yeast introns contain three highly conserved sequences which are known to be required for splicing of pre‐mRNA. Using in vitro mutagenesis, we have synthesized seven point mutations at five different sites in these signals in the yeast actin intron. The mutant introns were then inserted into each of three constructs, which allowed us to assess the consequences both in vivo and in vitro. In virtually every case, we found the efficiency of splicing to be significantly depressed; mature mRNA levels in vivo ranged from 0 to 47% of wild‐type. Surprisingly, the tightest mutations were not necessarily at the sites of nucleolytic cleavage and branch formation; these nucleotides are thus highly preferred, but are not absolutely necessary. Moreover, while particular nucleotides are specifically required for the final step in splicing, i.e. 3′ cleavage and exon ligation, the predominant consequence of mutation within the conserved signals appears to be the inhibition of assembly of the splicing complex.

Activity of axillary meristems dictates the architecture of both vegetative and reproductive parts of a plant. In Arabidopsis thaliana, a model eudicot species, the transcription factor LFY confers a floral fate to new meristems arising from the periphery of the reproductive shoot apex. Diverse orthologous LFY genes regulate vegetative-to-reproductive phase transition when expressed in Arabidopsis, a property not shared by RFL, the homolog in the agronomically important grass, rice. We have characterized RFL by knockdown of its expression and by its ectopic overexpression in transgenic rice. We find that reduction in RFL expression causes a dramatic delay in transition to flowering, with the extreme phenotype being no flowering. Conversely, RFL overexpression triggers precocious flowering. In these transgenics, the expression levels of known flowering time genes reveal RFL as a regulator of OsSOC1 (OsMADS50), an activator of flowering. Aside from facilitating a transition of the main growth axis to an inflorescence meristem, RFL expression status affects vegetative axillary meristems and therefore regulates tillering. The unique spatially and temporally regulated RFL expression during the development of vegetative axillary bud (tiller) primordia and inflorescence branch primordia is therefore required to produce tillers and panicle branches, respectively. Our data provide mechanistic insights into a unique role for RFL in determining the typical rice plant architecture by regulating distinct downstream pathways. These results offer a means to alter rice flowering time and plant architecture by manipulating RFL-mediated pathways.axillary meristem ͉ inflorescence branching ͉ flowering transition ͉ tillering A rabidopsis thaliana LFY and its homologs encode an evolutionarily conserved land plant-specific transcription factor. Early studies on the expression pattern and phenotypes of loss-of-function mutations in LFY and FLO, homologs in two dicots A. thaliana and Antirrhinum majus, showed them to confer a floral fate to new meristems arising on the flanks of the shoot apex (1, 2). LFY homologs from species as diverse as gymnosperms, primitive land plants, and from many angiosperms retain the ability to at least partially complement Arabidopsis lfy mutants (3). These data show activation of floral meristem fate to be a conserved LFY function. Protein domains recognizable in all LFY homologs are an N-terminal proline-rich domain and a C-terminal domain; substitutions in these largely conserved DNA-binding domains are suggested to contribute to its potentially divergent functions (3). In fact, mutations in some LFY homologs show additional developmental roles (e.g., compound leaf development in pea and cell division in moss) (4, 5).Unlike the simple inflorescence of Arabidopsis, grass inflorescences are striking in the multiple kinds of branch meristems made from the apical inflorescence meristem. In rice upon transition to reproductive phase, the vegetative apical meristem transforms to an inflorescence meristem. The...

MADS-domain-containing transcription factors play diverse roles in plant development. The prototypic members of this gene family are the floral organ identity genes of the model dicotyledonous plant, Arabidopsis thaliana. Sequence relatedness and function ascribe them to AP1/AGL9, AG, AP3 and PI gene groups. The rice MADS-box gene, OsMADS1, is a member of the AP1/ AGL9 sub-group. Tomato and Petunia members of this sub-group specify floral meristem identity and control organ development in three inner whorls. Reported here are phylogenetic analyses that show OsMADS1 to form a distinct clade within the AGL9 gene family. This sub-group currently has only three other monocot genes. We have studied the expression pattern of OsMADS1 and determined the consequences of its ectopic expression in transgenic rice plants. OsMADS1 is not expressed during panicle branching; earliest expression is in spikelet meristems where it is excluded from the outer rudimentary/sterile glumes. During organogenesis, OsMADS1 expression is confined to the lemma and palea, with weak expression in the carpel. Ectopic OsMADS1 expression results in stunted panicles with irregularly positioned branches and spikelets. Additionally, in spikelets, the outer rudimentary glumes are transformed to lemma/palea-like organs. Together, these data suggest a distinct role for OsMADS1 and its monocot relatives in assigning lemma/palea identity.