Response ofBrassica napus L. Microspore-derived embryos to exogenous abscisic acid and desiccation

Summary Microspore-derived embryos fromBrassica napus cv. Topas (low erucic acid) and Reston (high erucic acid) were subjected to treatment with abscisic acid (ABA) during late-stage embryo development and then dried under controlled relative humidities to mature dry seed levels of moisture. Exogenously medium-supplied ABA arrested growth and development, reduced moisture content, increased total fatty acids on a dry weight basis, and stimulated systhesis of proteins in microspore-derived embryos. ABA also resulted in a higher proportion of 22∶1 in cv. Reston (high 22∶1) and increased the level of fatty acid unsaturation in cv. Topas (low 22∶1). The accumulation of two proteins that co-migrated with cruciferin and napin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gels were also promoted by exposure to ABA, and the degree of accumulation was dependent on the concentration and time of application of ABA. Controlled desiccation of microspore embryos, used to simulate normal maturation and dehydration of zygotic embryos during seed development, did not seem to cause an increase of either storage proteins, total fatty acids, or 22∶1 (in cv. Reston), suggesting that dehydration is not a prerequisite for these processes, at least in culturedBrassica embryos.     

Comprehensive hormone profiling in developing Arabidopsis seeds: examination of the site of ABA biosynthesis, ABA transport and hormone interactions

ABA plays important roles in many aspects of seed development, including accumulation of storage compounds, acquisition of desiccation tolerance, induction of seed dormancy and suppression of precocious germination. Quantification of ABA in the F(1) and F(2) populations originated from crosses between the wild type and an ABA-deficient mutant aba2-2 demonstrated that ABA was synthesized in both maternal and zygotic tissues during seed development. In the absence of zygotic ABA, ABA synthesized in maternal tissues was translocated into the embryos and partially induced seed dormancy. We also analyzed the levels of ABA metabolites, gibberellins, IAA, cytokinins, jasmonates and salicylic acid (SA) in the developing seeds of the wild type and aba2-2. ABA metabolites accumulated differentially in the silique and seed tissues during development. Endogenous levels of SA were elevated in aba2-2 in the later developmental stages, whereas that of IAA was reduced compared with the wild type. These data suggest that ABA metabolism depends on developmental stages and tissues, and that ABA interacts with other hormones to regulate seed developmental processes.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Acquisition of Desiccation Tolerance and Longevity in Seeds of Arabidopsis thaliana (A Comparative Study Using Abscisic Acid-Insensitive abi3 Mutants)

Two new abscisic acid (ABA)-insensitive mutants of Arabidopsis thaliana affected in the abi3 locus are described. These new mutants are severely ABA insensitive. Like the earlier described abi3-1 and the ABA-deficient and -insensitive double mutant aba,abi3, these new mutants vary in the extent of ABA-correlated physiological responses. Mutant seeds fail to degrade chlorophyll during maturation and show no dormancy, and desiccation tolerance and longevity are poorly developed. Carbohydrate accumulation as well as synthesis of LEA or RAB proteins are often suggested to be essential for acquisition of desiccation tolerance. In this work two points are demonstrated. (a) Accumulation of carbohydrates as such does not correlate with acquisition of desiccation tolerance or longevity. It is suggested that a low ratio of mono- to oligosac-charides rather than the absolute amount of carbohydrates controls seed longevity or stability to desiccation tolerance. (b) Synthesis of a few assorted proteins, which is responsive to ABA in the later part of seed maturation, is not correlated with desiccation tolerance or longevity.     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity     

Abscisic Acid and the maturation of cacao embryos in vitro

Abscisic acid (ABA) was tested for its ability to affect development of immature zygotic embryos of cacao (Theobroma cacao) in vitro, by adding exogenous ABA, fluridone, or mefluidide to cultured embryos. Endogenous ABA levels, measured by enzyme-linked immunosorbent assay, were increased by exogenous ABA or by culture on sucrose increasing to 21%, and were decreased by fluridone and, to a lesser extent, by mefluidide. The effects of these on maturation were measured as effects on anthocyanins, lipids, and fatty acid saturation, all of which increase with maturation of the cacao embryo. Maturation was stimulated by increasing sucrose and, to a lesser degree, the addition of ABA, but decreasing endogenous ABA by treating with fluridone significantly inhibited all maturation parameters. Although desiccation tolerance does not develop in cacao embryos, these results suggest that ABA and sucrose are both needed for the initiation of events associated with maturation in vitro.     

Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane

Embryogenic calli of sugarcane (Saccharum sp. hybrid, clone CP52-43), with somatic embryos in the late scutelar stage, were subjected to different treatments for increasing embryo tolerance to desiccation. The medium was supplemented with abscisic acid (ABA) (3.8 μM), jasmonic acid (JA) (4.7 μM) or a combination of them. A control treatment without growth regulators was also included. The embryos were encapsulated in alginate beads and dehydrated or not in sucrose (0.5 M). Thereafter, they were further dehydrated in chambers containing silicagel until the beads reached either 60% or 30% of water content (WC). Survival of encapsulated-dehydrated embryos was achieved only in the control and ABA treatment. ABA induced an increase in protein, polyamines, free proline levels and starch levels as a response to desiccation tolerance. JA treatment showed the lowest protein and polyamines levels and increased the starch content almost two-fold compared to the ABA treatment. The JA treatment induced high levels of 4-methylcatechol and the lowest levels of gallic acid. However, the ABA treatment increased gallic acid and p-coumaric acid content in the induction medium. Some differences were found in growth regulator free-medium in relation to the induction medium. JA is not effective in these desiccation processes. The mechanisms by which these two plant growth regulators act on the induction of tolerance to stress are presumably different. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of desiccation tolerance in microspore-derived embryos ofBrassica napus

Summary A method was developed to induce desiccation tolerance in microspore-derived embryos ofBrassica napus. Treatment of 14- to 20-day-old embryos with 1×10^−4 M abscisic acid, in the light, induced tolerance to slow desiccation over a 6-day period. Under these conditions 88 to 100% of embryos of the five cultivars tested survived (as measured by moisture uptake, greening, and growth of the shoot and root meristem) after storage for 1 wk at tissue water content levels of less than 20%. The response was found to be dependent on the abscisic acid concentration in the culture medium and time of exposure of the embryos to the abscisic acid-containing medium, with exposure times of as little as 1 day having a beneficial effect. Exposure times to abscisic acid (ABA) of 5–7 days resulted in the highest survival rates. Embryo age and size at the time of ABA exposure also affected the subsequent survival and development of embryos, with older and larger embryos exhibiting the best responses.     

Desiccation tolerance in developing soybean seeds: The role of stress proteins

The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of certain "late embryogenesis abundant" (LEA) proteins suggests that these proteins reduce desiccation-induced cellular damage. The aim of the present work was to test this hypothesis. Exogenous abscisic acid (ABA) was used to elevate the level of heal-soluble LEA-like proteins in axes from immature (30 days after flowering: mid-development) seeds of soybean ( Glycine max [L.] Merrill cv. Chippewa 64). As the LEA-like proteins accumulated in response to ABA, the leakage of all elements after desiccation and subsequent rehydration markedly declined. Both LEA-like protein accumulation and the decline in desiccation-induced electrolyte leakage were apparently dependent on the presence of ABA. Both effects of ABA were inhibited by cycloheximide. Light microscopy revealed a marked effect of the ABA on cellular integrity following desiccation. Osmotic stress also caused a decrease in desiccation-induced electrolyte leakage and stimulated the accumulation of LEA-like proteins. Our data are consistent with the hypothesis that the LEA-like proteins contribute to the increase in desiccation tolerance in response to ABA, and are consistent with a general protective role for these proteins in desiccation tolerance.     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.