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The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed.

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.

Journal Article↗

Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

Fatty Acid Desaturases↗

EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis.

BACKGROUND: Plant germlines arise late in development from archesporial initials in the L2 layer of the anther and ovule primordia. These cells generate a radially symmetrical array of tissues that, in the Arabidopsis anther, comprises a core of sporogenous cells (meiocytes) and the enveloping tapetum, middle cell, and endothecium layers. The putative transcription factor NZZ/SPL is required for the specification of archesporial cells, but nothing is known of how their number is regulated, or what controls cell fate in the lineages they generate. Here, we report detailed characterization of extra sporogenous cells (exs), a male sterile mutant that generates extra meiocytes but lacks tapetal and middle cell layers. RESULTS: We identified the EXS locus by map-based cloning and found it to encode a putative LRR receptor kinase. In the anther, an increased number of L2 layer cells assume an archesporial fate and divide to generate a larger number of sporogenous cells. In seeds, the exs mutation results in smaller embryonic cells, delayed embryo development, and smaller mature embryos. Consistent with the observed phenotype, EXS is expressed in the inflorescence meristem, floral apices, anthers, and in developing seeds. CONCLUSIONS: EXS regulates the number of cells that divide in the L2 layer of the anther, and thus the number of functional male archesporial initials. In the young seed, EXS affects cell size in the embryo and the rate at which it develops. The apparently contrasting roles of EXS in the anther and embryo suggest that signaling through the EXS receptor kinase is a feature of a number of regulatory pathways in Arabidopsis.

Amino Acid Sequence↗

Structural and histochemical changes during seed development of Brassica macrocarpa Guss.

During seed formation of Brassica macrocarpa the development of the embryo precedes that of the integuments; structural changes and histochemical changes are associated. Esterases, acid phosphatases, phenols and starch follow a sigmoid pattern, increasing during embryogenesis and decreasing during seed maturation. In the mature seed, esterase activity is localized in the embryo and in the cells of the mucilaginous, aleuronic and hyaline layers. Acid phosphatases are present in the mucilaginous cells, mainly in the column, the cell walls delimiting intercellular spaces of the cortical cylinder and the adhesion areas of the cotyledons. Phenols are scanty in the root apex, mucilaginous cells and the palisade layer, and abundant in the pigmented layer. Starch is absent in ripe seeds which have lipid and protein reserves. The major classes of storage proteins have molecular weights of 21, 22, 27 and 30 KD and accumulate in the late stages prior to complete drying. Esterases and acid phosphatases in mucilaginous cells of the seed integument suggest that these enzymes are involved in hydrolytic processes occurring prior to germination and that mucilages have a metabolic function in seed-soil interactions.

Brassica↗

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION.

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C(20) and C(22) fatty acids, which contain an unusual Delta5 double bond. When [1-(14)C]acetate was incubated with developing seed slices, (14)C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [(14)C]acetate and [(14)C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[(14)C]11-eicosenoate, cis-13-[(14)C]docosenoate, and cis-5,cis-13-[(14)C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C(20) and C(22) acyl species. The Delta5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-(14)C-labeled free fatty acids. Using [1-(14)C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.

Journal Article↗

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Acetyltransferases↗

The phosphorylation state of the wheat translation initiation factors eIF4B, eIF4A, and eIF2 is differentially regulated during seed development and germination.

The translation initiation factors (eIF) 4B and eIF2 are phosphoproteins whose phosphorylation state differs between mature seed and leaves. We examined the isoforms of eIF4B and the alpha and beta subunits of eIF2 during the development and germination of wheat seed to determine whether the differences in their phosphorylation state are because of tissue-specific regulation or occur concomitant with changes in protein synthetic activity during development. eIF2alpha underwent phosphorylation through several intermediate isoforms that correlated with the increase and subsequent reduction in protein synthetic activity characteristic of seed development. eIF2beta and eIF4B, present as highly phosphorylated isoforms during early seed development, underwent dephosphorylation during late development. eIF4B was rapidly phosphorylated within 20 h of germination, whereas eIF2alpha did not undergo dephosphorylation until 48-60 h of growth. A third factor, eIF4A, was predominantly nonphosphorylated throughout most of seed development and germination. These observations suggest that the phosphorylation state of eIF2alpha, eIF2beta, and eIF4B is developmentally regulated in a way that correlates with the changes in protein synthetic activity but that some differences were also observed.

Electrophoresis, Gel, Two-Dimensional↗

Seed coat microsculpturing changes during seed development in diploid and amphidiploid Brassica species.

BACKGROUND AND AIMS: Seed coat morphology is known to be an excellent character for taxonomic and evolutionary studies, thus understanding its structure and development has been an important goal for biologists. This research aimed to identify the developmental differences of seed coats between amphidiploids and their putative parents in Brassica. METHODS: Scanning electron microscope (SEM) studies were carried out on six species (12 accessions), three amphidiploids and their three diploid parents. KEY RESULTS: Twelve types of basic ornamentation patterns were recognized during the whole developmental process of the seed coat. Six types of seed coat patterns appeared in three accessions of Brassica rapa, five types in B. oleracea, B. nigra and B. carinata, seven types in B. napus, and eight types in B. juncea. There was less difference among seed coat patterns of the three accessions of B. rapa. The reticulate and blister types were two of the most common patterns during the development of seeds in the six species, the blister-pimple and the pimple-foveate patterns were characteristic of B. rapa, and the ruminate of B. oleracea and B. nigra. The development of seed coat pattern in amphidiploids varied complicatedly. Some accessions showed intermediate patterns between the two putative parents, while others resembled only one of the two parents. CONCLUSIONS: The variation in the patterns of seed coat development could be used to provide a new and more effective way to analyse the close relationship among amphidiploids and their ancestral parents.

Brassica↗

The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice.

Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation.

Amino Acid Sequence↗

Expression of two genes encoding gibberellin 2- and 3-oxidases in developing seeds of Phaseolus coccineus.

We have isolated PcGA3ox1, a cDNA clone from developing runner bean (Phaseolus coccineus) seeds that shows significant amino acid homology with the gibberellin (GA) 3-oxidases. A recombinant fusion protein of PcGA3ox1 converted GA20 and GA9 to GA1 and GA4, respectively. In situ hybridization results showed that transcripts of this gene accumulate specifically within the suspensor of globular-stage embryos. PcGA3ox1 mRNA begins to accumulate in the epidermal cells of the embryo proper and is also detectable in the endosperm during the transition from globular- to heart-stage embryos. PcGA3ox1 transcripts were localized exclusively in the cotyledons from the early cotyledonary stage up to the cotyledonary stage. Transcripts of the previously cloned GA 2-oxidase (PcGA2ox1) from developing seeds of runner bean were found primarily within the suspensor neck region from the late globular stage up to the heart stage. PcGA2ox1 mRNA was detectable in the whole suspensor from the early cotyledonary stage, and was found in the inner layer of integuments at the cotyledonary stage. Soluble enzyme preparations made from suspensors and embryos at two stages of embryogenesis (the heart and cotyledonary stages) were incubated with [14C]GA20 and [14C]GA1. Only young suspensor preparations converted GA20 to GA1 and GA5. Both suspensor preparations converted GA1 to GA8. Both embryo preparations converted GA20 to GA1, but were unable to convert GA1 to GA8.

Cell-Free System↗

Developing Seeds of Ricinus communis L., When Detached and Maintained in an Atmosphere of High Relative Humidity, Switch to a Germinative Mode without the Requirement for Complete Desiccation.

Immature seeds of castor bean (Ricinus communis) removed from the capsule at 25 to 40 days after pollination (25-40 DAP) and placed in an atmosphere of high relative humidity undergo limited water loss, and germinate upon subsequent return to full hydration. This switch from a developmental to a germinative/growth mode at 40 DAP is reflected in a change in the types of proteins being synthesized in the endosperm; after partial drying, developmental protein synthesis ceases and germinative/growth-related proteins are produced. The nature and timing of these protein synthetic changes elicited upon imbibition are identical to those following premature desiccation/rehydration of 30 and 40 DAP seeds and upon imbibition of the mature dry seed. Enzymes involved in postgerminative reserve mobilization (l-leucyl-beta-naphthylamidase and isocitrate lyase) are induced upon imbibition, following partial drying at 40 DAP, to levels attained in the endosperms of germinated mature, and prematurely dried/rehydrated, seeds. The changes in protein synthesis resulting from partial drying are effected at the transcriptional and post-transcriptional level. Upon return to full hydration some new (i.e. germination and growth-related) mRNAs are synthesized, while others (associated with development) present in the partially dried endosperm decline. Thus developing seeds of castor bean do not have to experience substantial (whole seed) water loss to acquire the ability to germinate and grow upon subsequent imbibition. Seed detachment from the mother plant alone is not sufficient to elicit a switch to germination and growth processes. However, the length of time of detachment from the mother plant, in combination with some water loss may interact to elicit the "switch" from development to germination.

Journal Article↗

Temporal and spatial patterns of accumulation of the transcript of Myo-inositol-1-phosphate synthase and phytin-containing particles during seed development in rice.

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

Amino Acid Sequence↗

Gibberellins are required for seed development and pollen tube growth in Arabidopsis.

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.

Arabidopsis↗

Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains.

The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic profiling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the first time temporal and spatial maps of O2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained well-supplied with O2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, specific changes in the metabolite profiles (glycolysis and citrate cycle), and channelling of metabolic fluxes towards storage (increase of starch accumulation rate). These findings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level reflects the metabolic state of storage tissue. On the basis of these findings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.

Adenosine Triphosphate↗

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds.

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.

Arabidopsis↗

Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia.

Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation. Among these events are cell growth and differentiation and cellular metabolism. Casein kinase I (CKI) and casein kinase II (CKII) are involved in the phosphorylation of several substrates. Endogenous protein phosphorylation and casein kinase activity were investigated in the megagametophyte of the native Brazilian conifer Araucaria angustifolia, during seed development. It was observed that a number of different polypeptides are phosphorylated in vitro in the three megagametophyte stages of development tested (from globular, cotyledonary and mature embryos, respectively) and the phosphate was incorporated mainly in serine residues. The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the megagametophyte. Also, the results obtained in experiments with CKII inhibitor, GTP as phosphate donor, RNA hybridizations, and in-gel kinase assays indicate the presence of CKII in the A. angustifolia megagametophyte.

Casein Kinase II↗

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein.

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [(3)H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.

Journal Article↗

Coordinated accumulation of (+)-delta-cadinene synthase mRNAs and gossypol in developing seeds of Gossypium hirsutum and a new member of the cad1 family from G. arboreum.

A new member of the (+)-delta-cadinene synthase (CAD1) family was isolated from a Gossypium arboreum cDNA library. This cDNA encodes a protein that showed 97.3%, 96.9%, and 79.2% sequence identities with the proteins encoded by previously isolated cDNAs of cad1-C1, cad1-C14, and cad1-A, respectively. It may be grouped into the cad1-C subfamily as cad1-C2. Seeds of a glanded cotton cultivar, G. hirsutum cv. Sumian-6, were collected at different intervals during maturation, and the cad1 mRNA levels were analyzed by quantitative RT-PCR. The transcripts could be detected in seeds of 27 DPA (days postanthesis) and increased dramatically along with the seed maturation, which coordinated with an increase in sesquiterpene cyclase activities and subsequently the accumulation of gossypol. The transcription level detected with primers specific to cad1-C (including at least C1, C14, and cdn1) was higher than that detected with primers specific to cad1-A, and mRNA was detected also with cad1-C2-specific primers. This investigation indicates that, in developing seeds of the glanded cotton cultivar, genes of both the CAD1-C and CAD1-A subfamilies are expressed and there is an active biosynthesis of cadinene-type sesquiterpenes.

Amino Acid Sequence↗