The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development

Abscisic acid and osmoticum maintain maturation and proteinsynthesis of developing alfalfa embryos, individually and incombination. The in situ environment of developing alfalfa zygoticembryos is rich in ABA and low in osmotic potential. When ABAsynthesis was inhibited by treating the pods with fluridoneat an early stage of development, the seeds which subsequentlydeveloped contained low amounts of ABA, but had a similar osmoticpotential as untreated control seeds. The reduced ABA in seedsfrom fluridone-treated pods did not change the morphology exceptthe colour of seeds, nor did it induce viviparous germinationor affect storage protein synthesis. However, two nonstorageproteins which were synthesized in control seeds during earlyto mid-development were absent from fluridone-treated seeds.Control seeds containing these two proteins were desiccation-tolerant,whereas the fluridone-treated seeds which lacked them were desiccation-intolerant,at least until the deposition of storage proteins was nearlycomplete. Culture of isolated embryos on nutrient medium inducedgermination and curtailed storage protein synthesis in the embryos.Addition of either ABA or osmoticum to the nutrient medium preventedgermination and maintained storage protein synthesis. When fluridonewas added along with osmoticum, germination occurred, but storageprotein synthesis was maintained. Key words: Embryogenesis, Medicago sativa L., alfalfa, ABA, osmotic potential, fluridone, desiccation, storage protein synthesis     

Rapid and Reliable Differential Display from Minute Amounts of Tissue: Mass Cloning and Characterization of Differentially Expressed Genes from Loblolly Pine Embryos

Performing RNA differential display analysis on small tissue samples is difficult since much RNA, the initial template for the reaction, is lost during conventional isolation procedures. We have developed a rapid method which employs oligo-dT beads to capture mRNA from cell lysates. Subsequent reactions are primed directly from the beads, thus RT and PCR reactions can be completed within a few hours of tissue harvest. This approach allows us to perform differential display on a single pine embryo. We describe strategies for distinguishing classes of co-migrating bands excised from differential display gels and outline a PCR-based method for confirming differential expression of large numbers of cloned bands in cases where RNA quantities are limiting.     

Co-regulation Of ear growth and internode elongation in corn

Ear is the harvest part of corn (Zea mays L.) and we are interested in studying its growth and development in our effort in corn yield improvement. In our current study, we examined the relationship between ear growth and internode characteristics using different approaches. Correlations between stem growth rate and number of ears per plant (prolificacy) were assessed among several genotypes. Internode elongation of 2 genotypes was modified by plant hormones and by population density manipulations. Among the 7 genotypes examined that have different prolificacy levels, there was a general correlation of slower stem elongation at middle growth stages and larger ear number. When the internode elongation was enhanced by application of gibberellic acid (GA), ear growth was suppressed; and when a GA synthesis inhibitor uniconazole was applied at early stages, internode length was reduced and ear growth was promoted in terms of both ear size and visible ear number at silking stage. Higher population density caused longer internodes and fewer ears per plant and the effect of lower density was the opposite. Our results suggested that internode elongation in the middle section of corn plants was linked to suppression of ear development in corn.     

Roles of F-box proteins in plant hormone responses

The F-box protein is an important component of the E3 ubiquitin ligase Skpl-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. The F-box proteins contain a signature F-box motif at their amino-terminus and some protein-protein interaction motifs at their carboxyterminus, such as Trp-Asp repeats or leucine rich repeats. Many F-box proteins have been identified to be involved in plant hormone response as receptors or important medial components. These breakthrough findings shed light on our current understanding of the structure and function of the various F-box proteins, their related plant hormone signaling pathways, and their roles in regulating plant development.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos,but only osmoticum maintains the synthesis of developmental proteins

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10^?5 M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.     

Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression

During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.     

The role of phytohormones in cotton fiber development

Since 1974, when Beasley and Ting discovered that fertilized ovules of cotton can be cultured in media supplemented with GA along with auxin, the effect of all types of phytohormones on fiber development has been widely studied. Many phytohormones, including GA, IAA, brassinosteroid (Br), ABA, ethylene (Et), and cytokinins (Ck), all have been demonstrated to play important roles during cotton fiber development. In recent years, the rapid development of genomic analysis and the accumulation of high-quality cotton ESTs allowed us to probe phytohormonal gene expression during fiber development. Many phytohormonal genes, including GA-, IAA-, ABA-, Br-, Et-, and Ck-related genes, participating in phytohormone biosynthesis pathways and signal transduction pathway accumulated in the process of cotton fiber development.