Soybean Seed Protein Genes Are Regulated Spatially during Embryogenesis

We used in situ hybridization to investigate Kunitz trypsin inhibitor gene expression programs at the cell level in soybean embryos and in transformed tobacco seeds. The major Kunitz trypsin inhibitor mRNA, designated as KTi3, is first detectable in a specific globular stage embryo region, and then becomes localized within the axis of heart, cotyledon, and maturation stage embryos. By contrast, a related Kunitz trypsin inhibitor mRNA class, designated as KTi1/2, is not detectable during early embryogenesis. Nor is the KTi1/2 mRNA detectable in the axis at later developmental stages. Outer perimeter cells of each cotyledon accumulate both KTi1/2 and KTi3 mRNAs early in maturation. These mRNAs accumulate progressively from the outside to inside of each cotyledon in a "wave-like" pattern as embryogenesis proceeds. A similar KTi3 mRNA localization pattern is observed in soybean somatic embryos and in transformed tobacco seeds. An unrelated mRNA, encoding [beta]-conglycinin storage protein, also accumulates in a wave-like pattern during soybean embryogenesis. Our results indicate that cell-specific differences in seed protein gene expression programs are established early in development, and that seed protein mRNAs accumulate in a precise cellular pattern during seed maturation. We also show that seed protein gene expression patterns are conserved at the cell level in embryos of distantly related plants, and that these patterns are established in the absence of non-embryonic tissues.     

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp 18.6 G2 and Ha hsp 17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp 17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25°C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42°C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp 17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

4-Hydroxybenzyl alcohol accumulates in flowers and developing fruits of carrot and inhibits seed formation

Somatic embryogenesis in carrot (Daucus carota) is autonomously inhibited by 4-hydroxybenzyl alcohol (4HBA), which is produced by embryogenic cells. Because somatic embryogenesis is used as a model of zygotic embryogenesis, we assayed for 4HBA in carrot seeds and analyzed the effect of 4HBA on seed formation to determine whether 4HBA is also produced during zygotic embryogenesis. HPLC analysis showed that 4HBA accumulated in flowers and immature and mature fruits, but not in vegetative tissues. The concentration of 4HBA was highest after flowering, when the zygote developed into the early globular-stage embryo. 4HBA accumulation then decreased with seed development. Exogenous application of 4HBA to immature carrot fruits inhibited seed formation. Many 4HBA-treated seeds did not include a mature embryo. These results indicate that the production and accumulation of 4HBA occurs during carrot seed development and that 4HBA has an inhibitory effect on carrot seed formation.     

Organ-Specific and Environmentally Regulated Expression of Two Abscisic Acid-Induced Genes of Tomato : Nucleotide Sequence and Analysis of the Corresponding cDNAs

The cDNAs, pLE4 and pLE25, represent mRNAs that accumulate in response to water deficit and elevated levels of endogenous abscisic acid in detached leaves of drought-stressed tomato (Lycopersicon esculentum Mill., cv Ailsa Craig) (A Cohen, EA Bray [1990] Planta 182: 27-33). DNA sequence analysis of pLE4 and pLE25 showed that the deduced polypeptides were 13.9 and 9.3 kilodaltons, respectively. Each polypeptide was hydrophilic, cysteine- and tryptophan-free, and found to be similar to previously identified proteins that accumulate during the late stages of embryogenesis. pLE4 and pLE25 mRNA accumulated in a similar organ-specific pattern in response to specific abiotic stresses. Yet, expression patterns of the corresponding genes in response to developmental cues were not similar. pLE25 mRNA accumulated to much higher levels in developing seeds than in drought-stressed vegetative organs. pLE4 mRNA accumulated predominantly in drought-stressed leaves. The similarities and differences in the accumulation characteristics of these two mRNAs indicates that more than one mechanism exists for the regulation of their corresponding genes.     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.