Late Heart Stage Research Articles

SYNAPTOTAGMIN 4 is expressed mainly in the phloem and participates in abiotic stress tolerance in Arabidopsis.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum-plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Retarded Embryo Development 1 (RED1) regulates embryo development, seed maturation and plant growth in Arabidopsis

Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation. Thus, understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement. In this study, we report the identification of Retarded Embryo Development 1 (RED1) gene in Arabidopsis, whose two independent T-DNA insertion mutant lines, SALK_085642 (red1-1) and SALK_022583 (red1-2), show a retarded embryo development phenotype. The embryogenesis process ceases at the late heart stage in red1-1 and at the bent-cotyledon stage in red1-2, respectively, resulting in seed abortion in both lines. The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants. Small red1-2 homozygous plants can be successfully rescued by culturing immature seeds, indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation. Consistent with this observation, red1-2 seeds accumulate less protein, and the expression of two late embryo development reporter transgenes, LEA::GUS and β-conglycinin::GUS, was significantly weak and started relatively late in the red1-2 mutant lines compared to the wild type. The RED1 gene encodes a plant specific novel protein that is localized in the nucleus. These results indicate that RED1 plays important roles in embryo development, seed maturation and plant growth.

MINISEED3 ( MINI3 ), a WRKY family gene, and HAIKU2 ( IKU2 ), a leucine-rich repeat ( LRR ) KINASE gene, are regulators of seed size in Arabidopsis

We have identified mutant alleles of two sporophytically acting genes, HAIKU2 (IKU2) and MINISEED3 (MINI3). Homozygotes of these alleles produce a small seed phenotype associated with reduced growth and early cellularization of the endosperm. This phenotype is similar to that described for another seed size gene, IKU1. MINI3 encodes WRKY10, a WRKY class transcription factor. MINI3 promoter::GUS fusions show the gene is expressed in pollen and in the developing endosperm from the two nuclei stage at approximately 12 hr postfertilization to endosperm cellularization at approximately 96 hr. MINI3 is also expressed in the globular embryo but not in the late heart stage of embryo development. The early endosperm expression of MINI3 is independent of its parent of origin. IKU2 encodes a leucine-rich repeat (LRR) KINASE (At3g19700). IKU2::GUS has a similar expression pattern to that of MINI3. The patterns of expression of the two genes and their similar phenotypes indicate they may operate in the same genetic pathway. Additionally, we found that both MINI3 and IKU2 showed decreased expression in the iku1-1 mutant. IKU2 expression was reduced in a mini3-1 background, whereas MINI3 expression was unaltered in the iku2-3 mutant. These data suggest the successive action of the three genes IKU1, IKU2, and MINI3 in the same pathway of seed development.

Seed development in two species of medifixed hairy Astragalus (Fabaceae)

Summary Anatomical aspects of ovule, embryo and endosperm development are described for the zygote to cotyledon embryo stages in A. demavendicus and A. latifolius . Nucellar cells disappear during an early stage of development. The inner integument differentiates into an endothelium and an exterior layer of cells at the zygote stage. The primary endosperm nucleus divides before the zygote nucleus, forming a nuclear endosperm. In A. latifolius the polar nuclei fuse immediately after cellularization of the embryo sac, whereas in A. demavendicus they remain separated until fertilization. Endosperm cellularization begins when the embryo has developed the early globular stage in A. latifolius and the late globular stage in A. demavendicus . The inner integument is degraded at early heart stage in A. latifolius and at late heart stage in A. demavendicus , then the outer integument adjoins the embryo sac. The outer integument consists of three different tissues at this stage. As expected, the studied species which both belong to the same section of Astragalus , show major similarities in seed development. The remarked differences should be considered as variations among the species of this section.

Characterization of a tissue-specific and developmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum).

As part of a search for seed coat-specific expressed genes in Pisum sativum cv. Finale by PCR-based methods, we identified and isolated a cDNA encoding a beta- 1,3-glucanase, designated PsGNS2. The deduced peptide sequence of PsGNS2 is similar to a subfamily of beta-1,3-glucanases, which is characterized by the presence of a long amino acid extension at the C-terminal end compared to the other beta-1,3-glucanases. PsGNS2 is expressed in young flowers and in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is not inducible by environmental stress or in response to fungal infection. In developing pea flowers the transcript is detectable in all four whirls. In the seed coat the expression is temporally and spatially regulated. High abundance of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the mid seed-filling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo nutrition. This tissue showed also less callose deposits than the other ones. The 5' genomic region of PsGNS2 was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-specific expression. Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed.

Medea: murder or mistrial?

Recent articles on the medea (mea) mutant of Arabidopsis have described the fascinating way in which mutant mea alleles lead to embryo abortion[1–4]: when mutant copies of mea genes are transmitted through the female gametophyte they cause lethal abnormalities in seed development that cannot be rescued by wild-type copies from pollen. MEDEA is also interesting in that although it is clearly a maternal-effect gene (because it is expressed before fertilization) it is also transcribed post-fertilization—but only from the maternally derived alleles, implying that the paternally derived alleles are silenced by gametic imprinting[2]. At the time of seed desiccation, when wild-type embryos are fully differentiated, mea embryos have only reached late heart stage. Strikingly, the mutant embryos are up to ten times larger than normal embryos at late heart stage, and contain extra cell layers. These features led Grossniklaus et al.[1]to conclude that the wild-type function of MEA is to restrict cell proliferation during embryogenesis. The mea phenotype has also been presented as being in accord with the parental conflict hypothesis of imprinting, which proposes that maternally derived genes are selected to limit the growth of offspring[3]. However, a combination of features of the mea mutant phenotype are inconsistent with this interpretation, and in conjunction with data from other sources suggest that the situation is not so clear-cut. Other commentators[4, 5]have not addressed this issue, but we feel it is worth raising.

Biphasic expression of a Vicia faba legumin B gene in developing seeds of transgenic tobacco

Summary We analysed the spatial and temporal expression of the Vicia faba legumin gene LeB4 in developing seeds of transgenic tobacco plants by means of in situ hybridization and immunohistostaining. The results indicate that the expression of the LeB4 gene is regulated in embryo and endosperm in a biphasic manner. Legumin accumulation starts during early seed development at low amounts in single cells of the integument. Accumulation proceeds successively in the proembryo, suspensor and endosperm reaching maximum levels in all cells of these organs around 10 to 12 DAP. With the beginning of the heart stage (around 13 DAP) legumin reserves disappear completely from all parts of the seed. The described early phase of legumin accumulation occurs in embryo and endosperm still before the onset of the cell expansion phase in a period of continuous mitotic activity. A second expression phase starts in embryo and endosperm around 18 DAP, as the embryo enters late torpedo stage. Within the embryo the accumulation is restricted to the parenchyma cells of cotyledons and hypocotyl, while the embryonic root and provascular tissue do not accumulate LeB4 protein. In addition, a low percentage ( V. faba for the legumin and vicilin genes during early embryogenesis (Panitz et al., 1995), but differs from that of tobacco 12S globulin genes, which are expressed in the embryo not before the late heart stage.

A new Arabidopsis nucleic-acid-binding protein gene is highly expressed in dividing cells during development.

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation. This results suggests an expression pattern correlated with dividing cells.

Embryogenesis of Vicia faba L.: Histodifferentiation in Relation to Starch and Storage Protein Synthesis

Summary Seed development of Vicia faba is described and divided into seven stages based on morphological and histochemical characteristics: the globular stage, the early and late heart stage and four stages covering early to late cotyledon development. Specific criteria of these stages allow their easy identification independent of growth conditions and thus provide necessary reference points for any developmental studies. The distribution of mitotic activity followed by cell elongation and endopolyploidisation during seed development suggests a developmental gradient expressed in a specific spatial and temporal pattern at two levels between individual seed organs and within single organs. The accumulation pattern of storage proteins legumin and vicilin and their mRNAs, analysed by immunohistostaining and in situ hybridisation and the pattern of starch accumulation are directly correlated with these gradients, i.e., storage function is related inversely to mitotic activity and directly to cell elongation and endopolyploidisation. All seed organs synthesize storage proteins. Thus the expression of vicilin and legumin genes is not embryo specific but seed specific. Mobilisation of transiently stored storage proteins in suspensor, seed coat and endosperm occurs together with the degradation of the respective cell layers during development in a temporally regulated manner.

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10 −6 to 10 −7 M elicited the same protein diversity and greater [ 35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10 −6 to 10 −7, M restored the protein content to that of freshly excised embryos.

Light and electron microscopy of embryo development in an annual × perennial Medicago species cross

Using light and transmission electron microscopy, we studied embryo development from 3 to 14 days after pollination in an interspecific Medicago cross (perennial M. sativa L., 2n = 4x = 32 × annual M. scutellata (L.) Mill, 2n = 4x = 32) to determine factors contributing to abortion in wide crosses. An intraspecific M. sativa cross and an M. sativa self-mating were used for control embryos. Nucellus and integumentary tapetum at the base of the embryo sac appear to be important sources of nutrition during the first 2 weeks of embryo development. Micrographic evidence supports the conclusions that maternal and embryonic tissue of interspecific ovules fail to carry out a timely sequence of metabolism involving lipid, starch, and nucellar crystals. Delayed breakdown of starch and lipid in the integumentary tapetum and nucellus is a probable factor in reduced development of coenocytic endosperm. At the late heart stage of embryo development, relative inactivity of dictyosomes and endoplasmic reticulum in hybrid ovules suggests failure of nutrient metabolism and transport in all nutritive tissue including the nucellus, integumentary tapetum, endosperm, and suspensor of the embryo. Early embryo development in Medicago appears to be a complex phenomenon requiring a high degree of coordination between anabolism and catabolism in both maternal and embryonic tissue.

Embryogeny of Phaseolus: the Role of the Suspensor

Summary Label 14 C-sucrose is administered to the developing embryos via the base of the excised pod, through the endosperm cavity of the seeds, and directly to the excised embryos. The results indicate that the suspensor is the uptake site during early stages of embryo development. When labeled solution is introduced into the endosperm cavity at the late heart stage of embryo development, radioactivity appears in the suspensor and the suspensor end of the embryo-proper. This labeling pattern is unexpected. One might have expected the cotyledonary half of the embryoproper to be more heavily labeled since it is nearer to the endosperm — the source of radioactivity. The uptake of 14 C-sucrose by the suspensor at the late heart stage is sensitive to dinitrophenol whereas the total radioactivity in the embryo-proper increases gradually with time and the uptake of 14 C-sucrose is not inhibited by the presence of dinitrophenol. The uptake pattern is changed at the mid-maturation stage. It appears that the cotyledons become the major uptake site for the maturing embryo. The results are discussed in relation to the current hypotheses of suspensor function.

Embryogeny ofPhaseolus coccineus: Growth and microanatomy

During early embryogeny, the development of the suspensor is rapid both in terms of size and fresh weight; structural differentiation can be observed as early as the proembryo stage with the formation of wall ingrowths. Ingrowths first appear in the outer wall of the suspensor cells adjacent to the integumentary tapetum, soon ingrowths begin to form in the inner suspensor cells as well. A basal-terminal gradation in nuclear size exists, with the largest nuclei in the basal suspensor cells. Cytologically, the suspensor cells appear to be very active, especially when the embryo reaches heart stage. Initially, the development of the embryo proper lags behind the suspensor, but its size and fresh weight increase rapidly as development proceeds. The volume of the liquid endosperm rises most rapidly during the late heart stage; and it is absorbed soon after. A cellular endospermic sheath surrounds the embryo, separating it from the liquid endosperm. Structural differentiation also occurs in the cellular endosperm cells with the formation of wall ingrowths in those cells that abut directly onto the integumentary tapetum. Both the suspensor and the cellular endosperm appear to remain active through the maturation of the seed. Storage bodies are formed in the cotyledons as well as in the embryonic axis. In the suspensor and the cellular endosperm, starch grains and lipid bodies can be found at the maturation stage.

DEVELOPMENT OF SELF AND HYBRID DATURA EMBRYOS IN ARTIFICIAL CULTURE

DATURA INVESTIGATIONS in recent years have included growth of numerous interspecific hybrid embryos in artificial culture. Results obtained with embryos from different species combinations indicated that such embryos were unlike in their response to the nutrient solutions used. The following experiments were une MIcLean, 1946). Seitz-filtered malt extract added to the culture medium was found to promote embryo growth and was substituted for cocoanut milk. MATERIALS AND METHODS.-Seed development of the embryos used in the present investigations has been previously described (Sanders, 1948). They are self embryos of Datura discolor, D. inoxia, D. metel and D. stramonium; hybrid embryos from the compatible cross D. stramonium X D. discolor; and hybrid embryos from five incompatible crosses in which relatively numerous arrested embryos are formed. The incompatible crosses are D. discolor X D. stramonium, D. inoxia X D. discolor, D. inoxia X D. metel, D. inoxia X D. stramonium, and D. metel X D. inoxia. Self embryos of these species grow well in culture when dissected at stages of development ranging from the late heart stage to the mature embryo. Proembryos 0.1 mm. in diameter occasionally grow in culture but as a rule they do not. Embryos for these experiments were, therefore, taken at approximately the early heart stage when there is a real possibility that the embryo will grow and also that it will be more susceptible to changes in the nutrient than at older periods of developmient. At 1 Received for publication June 16, 1949. Contributions from the Department of Botany, Smitlh College, New Series No. 36. The article is taken from a thesis written under the direction of Dr. A. F. Blakeslee and submitted to the faculty of Smith College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. this stage the top of the proembryo has flattened out, cotyledon primordia are just beginning to appear, and the longitudinal axis is about 0.2 mm. Slightly larger embryos were occasionally used because of the difficulty in obtaining the exact stage desired. Degree of embryo development at the time of dissection must be considered since it has been found that growth of embryos in culture varies with the stage of the embryos. Hybrid embryos from the compatible cross (D. stramonium X D. discolor) were also dissected at approximately the early heart stage though in some cases cotyledon differentiation was not evident. Embryos in this cross do not always differentiate normally so that such cases were probably examples either of the lack of normal differentiation or of the delay of differentiation. Hybrid embryos from incompatible crosses, due to the difficulty of obtaining a sufficient number for the experiments, were dissected at the age when the most embryos could be found, old enough for developing embryos to be seen, and young enough for the embryos to be still viable. This age varied with the cross (Sanders, 1948). Embryos were dissected out of the seeds under sterile conditions with the aid of a dissecting microscope and introduced into cotton-plugged vials containing culture media which had been sterilized by autoclaving at 15 lbs. pressure for 20 min. When malt was used in the nutrient, it was sterilized by Seitz-filtering and added to the vials after autoclaving. All nutrients to be compared were used in the same experiment to keep environmental factors affecting growth the same for all. Embryos were introduced into the vials along rows at right angles to the rows representing different nutrients in order to distribute embryos from the same capsule and to balance differences in size and vigor of the embryos among the nutrients. Vials were kept in an incubator at a constant temperature of 26?C. Growth has been expressed as the number of times the embryos increased in size during their days in culture. Size was determined by measuring the longitudinal axis of the embryo or of the largest outgrowth in the cases where multiple growth occurred. Measurements were made the dav of dissection and again after the prescribed number of days in culture. The growth value used is-final average size/original average size. Results are expressed as growth values or as the average of growth values from several experiments. Each experiment was done twice, occasionally three times, with ten embryos per nutrient so that the results are based on at least twenty vials per treatment. Data from individual experiments which were averaged together to obtain the growth values