The regulation of cotton embryogenesis has been addressed by measuring the abundance of 47 mRNAs in cotyledons from the late cotyledon stage through early germination. There are at least 11 distinct classes of coordinately expressed mRNAs. Their expression patterns appear to result from unique combinations of five temporal abundance components. These are associated with the cotyledon stage, the endogenous concentration of free abscisic acid, maturation (reserve accumulation), ovule abscission, and germination. This modularity suggests that only a few global regulatory factors orchestrate gene expression with many genes responding to several of them. Significant expression associated only with postabscission or free abscisic acid is restricted to that of the Lea mRNAs earlier suggested to be a component of the embryo's preparation for desiccation.
The major programs of gene epxression during late embryogenesis are the maturation or reserve accumulation program and, after ovule abscission, the postabscission program that is composed largely of Lea and LeaA mRNAs that probably encode desiccation protectants. There are diverse opinions about the developmental regulators of these programs. Several candidates are evaluated here by measuring, in cultured embryos, the accumulation kinetics of cloned mRNAs specifically expressed in the normal maturation, postabscission, or germination programs of cotton. Maturation-stage embryos both terminate the maturation program and induce the postabscission program after excision and culture, just as they do later in the plant after ovule abscission. However, they also induce simultaneously the germination program and thus are different from any normal stage of embryo development or germination. The developmental induction of the postabscission program in culture does not require exogenous abscisic acid, but its expression is enhanced by precocious desication or culture on abscisic acid or high osmoticum, probably by an environmentally responsive mechanism that normally operates during germination. Normal desiccation does not control any of these programs because the embryo acquires all of the characteristics of a mature embryo before it desiccates. These and other results suggest regulation of normal embryogenesis by a maternal maturation factor, a postabscission factor, and the postabscission program.
During seed formation, the embryo appears to be germinable as soon as cell division is completed; however, it continues development on the plant. This review describes the stages of development after cell division and provides a summary of important observations and recent use of molecular markers as they apply to the regulation of dicot seed formation. Genetic evidence suggests that abscisic acid may help initiate late embryogenesis, although no evidence firmly establishes that abscisic acid controls any other aspect of late dicot development. Previous studies utilizing cultured embryos have implicated abscisic acid and water potential as endogenous promoters of late embryogenesis and inhibitiors of germination. However, these embryo culture experiments have been misinterpreted. The experiments show that both immature and mature embryos respond to environmental water stress by expressing a developmental program that is normally induced in late embryogenesis by abscission of the vascular connection. This postabscission program probably prepares the embryo for its forthcoming desiccation during normal development and is predicted to be important in protecting the embryo from water stress during germination.
As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridizes to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were a nearly full-length aleurone/pericarp clone pB22E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcripts and contains two introns of 145 and 125 bp. Particle bombardment of barley aleurone with a B22EL8 promoter-GUS (B-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of the 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.
LEA proteins are a diverse group of probable desiccation protectants that are developmentally induced during the postabscission stage of late embryo development by ovule abscission and are environmentally induced, probably by a different mechanim(s), in cultured embryos by ABA, desiccation, and osmotic stress. Of the 18 Lea mRNAs cloned from cotton, mRNAs of the two related genes Lea4, a Group 1 Lea gene, and LeaA2 have the highest cross-hybridization with embryo mRNAs of other plants. A recent compilation of Group 1 LEA sequences confirms a very high conservation at the amino acid level. To define the relationship of the cotton Group 1 proteins encoded by Lea4 and LeaA2, and to help understand the basis of their differential expression during development, both alloalleles of both genes were sequenced. LeaA2-encoded proteins are very similar to the one encoded by Lea4, except that they contain a tandem duplication of a 20-amino acid sequence present only once in the Lea4-encoded protein.
The late embryogenesis-abundant (Lea genes, which are suggested to act as desiccation protectants during seed desiccation and in water-stressed seedlings, can be induced by abscisic acid (ABA) and various kinds of water-related stress. Using cotton Lea cDNA probes it was found that several of the Lea genes are conserved at the mRNA level in dicots and monocots. By screening a barley cDNA library with a cotton Lea D19 cDNA a family of three members was isolated. The putative B19 proteins have strong similarities to the Em protein in wheat and to LEA proteins from several dicots. However, the middle part of the B19 proteins consists of a 20-amino acid motif repeated three and four times in B19.3 and B19.4, respectively, but only once in B19.1. The gene products are strongly hydrophilic, the internal 20-amino acid motif being the most hydrophilic part. This motif is found once in cotton Lea D19 but is repeated twice in cotton Lea D132, indicating that the repeats are universal among monocot and dicot B19-like genes. The B19 genes are regulated similarly during embryo developoment, but to very different levels. In contrast, they are differentially regulated by ABA and various types of osmotic stress. In immature embryos all three genes are responsive to ABA and mannitol. However, B19.1 is also responsive to salt. Cold stress does not induce B19 mRNAs; only a stabilization of the transcript levels is seen. These results suggest that the responses to salt stress and exogenous ABA operate through different pathways.
Of the many Lea and water stress-related mRNAs that have been sequenced, all but cotton Lea12 and the desiccation-induced Craterostigma cDNA pcC27-45 encode proteins that are very hydrophilic. In cotton, only Lea5A and Lea14 are highly induced in mature leaves of water-stressed plants or in water-stressed, detached leaves. We report here that both of these genes encode proteins with significant hydropathic character and that Lea14 is a homolog of the gene encoding the Craterostigma desiccation-induced mRNA.
Cloned mRNAs identify three programs of gene expression in cotton (Gossypium hirsutum L.) embryos that are associated with the maturation (reserve accumulation) stage, the posTabscission stage, which is marked by expression of Late-embryogenesis-abundant (Lea) mRNAs, and germination (broadly defined as including all events through early postgerminative growth). In order to test if the regulation of these programs is the same in other dicotyledonous species, their expression was studied in normal and cultured maturation-stage, postabscission-stage, and mature embryo-stage embryos or seed of oilseed rape (Brassica napus L.), soybean (Glycine max [L.] Merr.), and tobacco (Nicotiana tobacum L.) using cotton and other cDNA probes. During postabscission, Lea mRNAs accumulated in all test species and were induced in earlier maturation-stage embryos by excision and culture on basal medium. Abscisic acid often enhanced this induction in the test species. Germination-specific mRNAs were induced in cultured maturation-state and postabscission-stage embryos of all test species. These results indicate that the regulation of embryonic and germination programs are similar in all dicotyledons tested. Because excised embryos simultaneously induced postabscission and germination programs, the effects of exogenous growth regulators and other factors on such embryos probably reflect stress responses of germinating mature embryos rather than the identity of endogenous regulators of embryogenesis.
In Ricinus communis L. (caster bean) endopserms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30-35 days after pollination, DAP) and peaked at 50 DAP, just prior to the onset of desiccation. Most of the Class I mRNAs declined substantially during desiccation itself; Class II mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, all Lea mRNAs abundant in the mature dry seed declined rapidly (within 5-24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding post-germinative proteins (GERM D91, D30 and D38) increased in the endosperm. The Lea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration these Lea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as 'dehydrins'; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not evident in prematurely dried endosperms. In contrast to the rapid loss of Lea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.
Positional cloning of mutated genes continues to be an essential method for identifying genes and their biological function. Mapping genes in Arabidopsis has changed dramatically since the introduction of PCR-based techniques such as Amplified Fragment Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS). Critical mapping parameters have been improved to accelerate mapping with the CAPS markers approach. Higher efficiency and throughput for large mapping populations are gained by a modified DNA extraction method, generating sufficiently pure DNA from a small amount of dry seeds instead of commonly used vegetative tissue. In order to enhance the reproducibility of PCR reactions, a faster and reduced PCR and restriction enzyme digestion protocol is combined with experimentally optimised PCR conditions for over 50 various CAPS primer pairs. Finally, the density of genetic markers is increased by providing the complete polymorphic information for all the CAPS markers for the widely-used Arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi), greatly enhancing the mapping possibilities with these ecotypes.
Dormant Arabidopsis seeds require stratification and light for germination. To study gene expression during establishment, maintenance and release of dormancy, various Arabidopsis ecotypes that are different in their degree of dormancy were investigated: three nsm mutants that lack the stratification-dependency, and the precocious germination and reduced dormancy of the abi3-1 mutant (insensitive to ABA). Genes examined by mRNA abundance include LEC1, FUS3 and ABI3, transcription factors that are major regulators of embryo development and, at least indirectly, play some role in the control of dormancy. Moreover, the late embryogenesis marker genes, AtEm1 and AtEm6, were examined in relation to the state of dormancy. The expression of LEC1, FUS3 and ABI3 mRNA is only marginally different during seed development in various strong or moderate dormancy wild types, nsm mutants and abi3-1. Therefore, it is unlikely that these transcription factors directly control the establishment of dormancy in Arabidopsis. Sole and various combinations of light, temperature, and after-ripening regimes that alter germination behaviour were examined to determine if the expression of ABI3, AtEm1 and AtEm6 mRNAs were correlated with dormancy-breaking processes. ABI3 expression is influenced by cold and light, in a similar way in both dormant and nondormant wild-type seeds. ABI3 transcript abundance in the nsm1 and nsm2 mutants is higher and in the nsm5-1 mutant is marginally lower than in wild-type seeds, but changes due to temperature and light factors are very similar to those that occur in wild-type seeds. The abundances of AtEm1 and AtEm6 mRNAs are equally affected by imbibition and cold temperature in mature and after-ripened seeds. The LEA transcript abundances for AtEm1 and AtEm6 are reduced in a common, ABI3-independent pathway.