Rowan S. McKibbin

Transcripts of Vp-1 homeologues are misspliced in modern wheat and ancestral species

The maize (Zea mays) Viviparous 1 (Vp1) transcription factor has been shown previously to be a major regulator of seed development, simultaneously activating embryo maturation and repressing germination. Hexaploid bread wheat (Triticum aestivum) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting (PHS), a phenomenon that is phenotypically similar to the maize vp1 mutation. Analysis of Vp-1 transcript structure in wheat embryos during grain development showed that each homeologue produces cytoplasmic mRNAs of different sizes. The majority of transcripts are spliced incorrectly, contain insertions of intron sequences or deletions of coding region, and do not have the capacity to encode full-length proteins. Several VP-1-related lower molecular weight protein species were present in wheat embryo nuclei. Embryos of a closely related tetraploid species (Triticum turgidum) and ancestral diploids also contained misspliced Vp-1 transcripts that were structurally similar or identical to those found in modern hexaploid wheat, which suggests that compromised structure and expression of Vp-1 transcripts in modern wheat are inherited from ancestral species. Developing embryos from transgenic wheat grains expressing the Avena fatua Vp1 gene showed enhanced responsiveness to applied abscisic acid compared with the control. In addition, ripening ears of transgenic plants were less susceptible to PHS. Our results suggest that missplicing of wheat Vp-1 genes contributes to susceptibility to PHS in modern hexaploid wheat varieties and identifies a possible route to increase resistance to this environmentally triggered disorder.

Genetic map locations for orthologous Vp1 genes in wheat and rice

Abstract Chromosome locations for gene orthologues of the dormancy-related maize transcription factor VIVIPAROUS-1, encoded by the Vp1 locus on maize chromosome 3, were determined in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) via linkage to markers on existing molecular maps using a cDNA of a wheat Vp1 orthologue as a probe in genomic Southern analyses. Vp1-orthologous loci were detected on the long arms of wheat chromosomes 3A, 3B and 3D [Xlars10 (taVp1) loci] and rice chromosome 1 (osVp1), in line with previous evidence of synteny between these regions of the rice and wheat genomes and chromosome 3 of maize. The wheat loci mapped some 30 cM from the centromeres and some 30 cM proximal to the red grain (R) loci that control seed colour and coat-imposed dormancy. This unequivocal, genetic separation of the Vp1 and R loci may offer an opportunity for improving resistance to pre-harvest sprouting in wheat by combining the coat-imposed dormancy associated with red seed colour and true embryo dormancy regulated by Vp1.

Molecular and genetic mechanisms regulating the transition from embryo development to germination

Abstract How do plants regulate development from embryogenesis through to germination? Mutational analysis of embryo maturation and germination in Arabidopsis and maize has provided a framework for dissecting the regulatory processes required for this transition. Genetic loci have been identified that are responsible for both repressing premature germination and simultaneously stimulating embryo development. Several of these loci have now been cloned, and an analysis of their molecular properties, in combination with analysis of their genetic interactions, is providing insights into how the transition between seed and seedling is regulated and coordinated.

Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco.

Lateral root formation was examined following the expression of a fission yeast mitotic regulator gene, cdc25, under the control of a tetracycline-inducible promoter in cultured roots of tobacco. Over expression of cdc25 in fission yeast results in premature cell division at a reduced cell size. Our aim was to examine whether cdc25 expression would affect cell size in the tobacco roots, and what effect this would have on lateral root morphogenesis. Transgene integration was confirmed by Southern blotting; it was inherited as a dominant Mendelian trait. Conditions for optimal expression, determined using plants transformed with gus under the control of the same promoter, were: addition of tetracycline (5 µg/ml) every 72 h, to cultured roots in Murashige-Skoog liquid medium in darkness at 27 °C. After the addition of tetracycline, cdc25 transcripts were detected using RT-PCR, initially after 48 h, and more strongly after 72 h. Appearance of cdc25 transcripts was followed by major changes in the roots. Compared with controls, lateral root primordia were initiated more frequently, were significantly smaller and comprised smaller cells at mitosis. However, cdc25 expression did not perturb normal development of the lateral roots. The data are consistent with cdc25 expression leading to a greater frequency of lateral root primordium formation and establishing a new threshold size for cell division in the primordia which was then maintained throughout subsequent development.

Genetic control mechanisms regulating the initiation of germination

Summary Genetic analysis of the transition from embryogenesis to germination has shown that these processes are under strict sequential, mutually exclusive, control. Understanding the regulation of this phase transition should provide important approaches to new technologies that can be usedto improve seed quality traits in crop plants. The developmental disorder pre-harvest sprouting (PHS) in cereals and pre-germination of seeds in the pods of oilseed rape are significant agronomic problems, that occur when seeds develop under cool moist environmental conditions. Phenotypically, pre-germination of seeds on the mother plant is similar to the severe maize and Arabidopsis mutations viviparous1 ( vp1 ), and abscisic acid insensitive3 ( abi3 ).The corresponding loci encode homologous transcription factors thatsimultaneously activate embryo maturation and repress germination. We have analysed gene expression programmes in wheat embryos under conditions that induce PHS, and have analysed in detail the structure and expression of wheat Vp1 homeologues. These analyses show that both maturation and germination genes are expressed simultaneously in embryos grown under cool moist conditions, and that the majority of VP1 transcripts expressed in the cytoplasm during normal embryo maturation are not correctly spliced. These results suggest that under perturbed environmental conditions wheat may not express enough functional VP1 activity to repress germination. This hypothesis is currently being tested using transgenic approaches. Repression of germination by ABI3 andother loci in Arabidopsis indicates that these factors interact with loci that enhance germination potential. Using a novel genetic screen to search for regulators of germination, we have identified the COMATOSE ( CTS ) locus. Genetic and physiological analyses show that CTS regulates germination potential by enhancing after ripening, sensitivity to gibberellins and pre-chilling, and by repressing the activities of loci that activate embryo maturation.

Use of comparative molecular genetics to study pre harvest sprouting in wheat

The use of model genetic systems in plant and animal studies has allowed a greatly increased understanding of how genomes regulate phenotype. Arabidopsis thaliana (arabidopsis) has proved to be an extremely useful model for plant molecular genetic studies and there are now many examples of important agricultural genes that have been identified using this species as a model. The discovery and investigation of a large number of arabidopsis mutants associated with seed development and germination indicates that resources are available, using comparative molecular genetics approaches, to identify candidate loci that may play important roles in the regulation and aetiology of pre-harvest sprouting (PHS). Specific examples are discussed including the ABSCISIC ACID INSENSITIVE3 (ABI3) and FUSCA3 (FUS3) loci, that encode proteins containing a highly conserved DNA binding domain originally identified in the maize Viviparous-1 (Vp-1) transcription factor. The potential use of this gene family for the manipulation of PHS, either through marker-assisted breeding or transgenic approaches is discussed.