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PubMed1979

The purification and characterization of ADP-glucose pyrophosphorylase A from developing maize seeds.

ADPglucose pyrophosphorylase A (ATP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) from developing maize (Zea mays) endosperm was purified 129 fold to apparent homogeneity. The molecular weight estimated by gel filtration and by polyacrylamide gel electrophoresis was 375 000 and 400 000, respectively. The preparation gave a single protein band after SDS-polyacrylamide gel electrophoresis suggesting a monomer mol. wt. of 96 000. It was concluded that ADPglucose pyrophosphorylase A in maize endosperm is a tetramer of four similar molecular weight subunits. Values for the Km for glucose 1-phosphate and ATP were 3.8 . 10(-5) and 1.8 . 10(-4) M, respectively (using the homogeneous preparation).

Adenosine Diphosphate Glucose↗
PubMed2001

The Rop GTPase switch controls multiple developmental processes in Arabidopsis.

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.

Arabidopsis↗
PubMed1999

Localization of peroxidase mRNAs in soybean seeds by in situ hybridization.

The soybean Ep gene encodes an anionic peroxidase enzyme that accumulates in large amounts in seed coat tissues. We have isolated a second peroxidase gene, Prx2, that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase isozyme that is far removed from Ep in peroxidase phylogeny. To determine the expression patterns for these two peroxidases in developing seeds, the abundance and localization of the Ep and Prx2 transcripts were compared by in situ hybridization. Results show the expression of Ep begins in a small number of cells flanking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. Nonetheless, the Ep-encoded enzyme accounts for virtually all of the peroxidase activity detected in mature seed coats. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subject to degradation during seed maturation.

Blotting, Northern↗
PubMed2000

Disturbance in the allocation of carbohydrates to regenerative organs in transgenic Nicotiana tabacum L.

Transgenic tobacco (Nicotiana tabacum L, cv. SR-1) expressing mannitol 1-phosphate dehydrogenase, MTLD, in chloroplasts and myo-inositol O-methyltransferase, IMT1, in the cytosol after crossing of lines which expressed these foreign genes separately has been analysed. Plants expressing both enzymes accumulated mannitol and D-ononitol in amounts comparable to those following single gene transfer and showed phenotypically normal growth during the vegetative stage. Induction of flowering for transgenovar and wild-type occurred at the same time, but during flowering the phenotype of the transformed plants changed. Compared to wild-type, transgenic plants were characterized by curled, smaller upper leaves and elongated stems during flowering; incomplete development of flower buds with shorter sepals and pedicels resulted in increased abortion. Flowers completing development were normal. The vegetative biomass of the transformed plants was slightly higher than that of wild-type. Concentrations of soluble sugars and potassium were lower than in wild-type only in the apical parts of the transgenic plants. Both enzymes, under control of the CaMV 35S promoter, promoted accumulation of mannitol and D-ononitol in the youngest leaves close to the vegetative meristem and in flowers, suggesting that their presence could signal lower sink demand leading to a decrease in carbon import to flowers and developing seed capsules. The interpretation here is that increases of inert carbohydrates in developing sinks interfere with metabolism, such as respiration or glycolysis. This interference may be less significant in source tissues during vegetative growth than in sink tissues during seed development.

Carbohydrate Metabolism↗
PubMed2001

Identification of genes specifically expressed in maternal and filial tissues of barley caryopses: a cDNA array analysis.

Developing seeds consist of genetically distinct maternal and filial tissues, whose interactions during development are largely unknown. To better understand the molecular physiology of developing seed tissues in barley, we created a high-density cDNA macroarray bearing 711 cDNA fragments from 691 clones representing at least 620 unique genes mainly derived from a cDNA library constructed with mRNA from the early stages of caryopsis development. This array has been used to compare gene expression patterns in maternal pericarp and filial embryo sac tissues of caryopses sampled 1-7 days after flowering (DAF). The profiles obtained for both tissues revealed that at least 26 genes in pericarp and 12 genes in embryo sac tissues were up-regulated by more than a factor of two during this period. RNAs expressed at high levels in the pericarp mainly encode enzymes involved in carbohydrate and lipid metabolism, but also include mRNA for a transcription factor related to FILAMENTOUS FLOWER (FIL). Genes preferentially expressed in the embryo sac are mainly related to degradation and/or processing of proteins or are involved in the process of starch accumulation, which begins in the seed at this time. Some of the most conspicuously regulated genes were studied in more detail by Northern analysis and in situ hybridization. The mRNA with the highest apparent signal intensity encodes a methionine synthase (MSY). MSY is highly expressed throughout the pericarp and to a lower extent in the transfer cell layer of the endosperm. Of special interest is a gene of unknown function because its high-level expression is restricted to the nucellar projection, the maternal transfer tissue of the caryopsis. This gene, represented by clone HY09L21, may play a central role in transport processes and thus in embryo growth.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2000

Maternal genotype influences pea seed size by controlling both mitotic activity during early embryogenesis and final endoreduplication level/cotyledon cell size in mature seed.

When reciprocal crosses are made between different pea genotypes, there is a strong maternal influence on mature seed size of the reciprocal hybrids, i.e. their dry weights are similar to that of seeds obtained from their maternal parents. Reciprocal crosses between pea varieties having very different mature seed sizes were used to investigate how the maternal genotype controls seed development and mature seed size. The differences in dry seed weight between genotypes and reciprocal hybrids reflected differences in both cotyledon cell number and mean cell volume, and the maternal control on the establishment of these two traits was investigated. Using flow cytometry, data relative to endoreduplication kinetics in cotyledons during the transition between the cell division phase and maturation were obtained. The appearance of nuclei having an 8C DNA content indicates the initiation of the endoreduplication phenomenon and thus the end of the cell division phase. It was shown that the duration of the cell division phase was the same in the reciprocal hybrids, its value being intermediate between those recorded for their maternal parents. This result indicates that the timing of development of the embryo is not under maternal control, but depends on its own genotype. Consequently, maternal genotype must influence the mitotic rate during the cell division phase to achieve differences in cell number found in the cotyledons of mature F1-reciprocal hybrids. The final level of endoreduplication in cotyledons of mature seeds was also investigated. This study showed that there is a close relationship (r2 = 0.919) between the endoreduplication level in mature cotyledons and seed dry weight or mean volume of cotyledon cells, suggesting that both maternal and non-maternal factors could control the number of endoreduplicating cycles in the cotyledons and, hypothetically, the cotyledon cell size.

Genotype↗
PubMed1978

6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L.

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl(2) stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP(+) were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.

Journal Article↗
PubMed2006

Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy.

The cleavage of 9-cis-epoxycarotenoids to xanthoxin, catalyzed by 9-cis-epoxycarotenoid dioxygenases, is considered to be the key regulatory step of abscisic acid (ABA) biosynthesis. In Arabidopsis, genes for these enzymes form a multigene family with nine members, only five of which are thought to be involved in ABA production. In contrast to the prominent function of AtNCED3 in stress responses, the physiological and developmental role of the other 9-cis-epoxycarotenoid dioxygenases (NCEDs) remain unknown. Our functional and expression analyses have revealed that AtNCED6 and AtNCED9 are required for ABA biosynthesis during seed development. Reverse genetic analysis showed that ABA levels were reduced in Atnced6 and Atnced9 mutant seeds. In addition, transgenic plants overexpressing the AtNCED6 gene overproduced ABA. In accordance with mutant phenotypes, both AtNCED6 and AtNCED9 exhibited seed-specific expression. Detailed cytological studies were carried out, either by using transcriptional fusions of the promoter with GUS and GFP reporter genes, or by in situ hybridization. Expression of AtNCED6 was observed exclusively in the endosperm during seed development, that of AtNCED9 in both embryo and endosperm at mid-development. In addition to reduced ABA levels, Atnced6 and Atnced9 mutant seeds were also resistant to paclobutrazol, a gibberellin biosynthesis inhibitor. Although seeds of single mutants were still dormant, reduced dormancy was observed in the Atnced6 Atnced9 double-mutant seeds. These demonstrate that ABA synthesized in both the endosperm and the embryo participates in the hormonal balance that controls seed dormancy and germination.

Abscisic Acid↗
PubMed1992

Structure and expression of the barley lipid transfer protein gene Ltp1.

We have characterized a gene (Ltp1) encoding a barley lipid transfer protein. Northern blot analysis showed that Ltp1 mRNA accumulates specifically in the aleurone layer of developing and germinating seeds. Southern blot analysis indicated that LTP1 protein is encoded by a single gene in barley. Sequence analysis of Ltp1 showed that it contains an open reading frame of 351 bp interrupted by a single intron of 133 bp. Transient expression assays indicated that 702 bp of the 5' upstream region of Ltp1 is sufficient to direct aleurone-specific expression during late seed development and early germination.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase from developing soybean seeds.

Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into alpha-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K(m) for its substrates, Lys and alpha-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1-13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys.

Amino Acid Sequence↗
PubMed1999

The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells.

Protection against desiccation-induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by-products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation-induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free-radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol-requiring anti-oxidants with one conserved cysteine residue (1-Cys), and displays in vitro anti-oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy-related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non-dormant seeds. Our data identify the location of this seed-specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C-terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.

Amino Acid Sequence↗
PubMed1992

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A.

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.

2S Albumins, Plant↗
PubMed1997

Suspensor-derived polyembryony caused by altered expression of valyl-tRNA synthetase in the twn2 mutant of Arabidopsis.

The twn2 mutant of Arabidopsis exhibits a defect in early embryogenesis where, following one or two divisions of the zygote, the decendents of the apical cell arrest. The basal cells that normally give rise to the suspensor proliferate abnormally, giving rise to multiple embryos. A high proportion of the seeds fail to develop viable embryos, and those that do, contain a high proportion of partially or completely duplicated embryos. The adult plants are smaller and less vigorous than the wild type and have a severely stunted root. The twn2-1 mutation, which is the only known allele, was caused by a T-DNA insertion in the 5' untranslated region of a putative valyl-tRNA synthetase gene, valRS. The insertion causes reduced transcription of the valRS gene in reproductive tissues and developing seeds but increased expression in leaves. Analysis of transcript initiation sites and the expression of promoter-reporter fusions in transgenic plants indicated that enhancer elements inside the first two introns interact with the border of the T-DNA to cause the altered pattern of expression of the valRS gene in the twn2 mutant. The phenotypic consequences of this unique mutation are interpreted in the context of a model, suggested by Vernon and Meinke [Vernon, D. M. & Meinke, D. W. (1994) Dev. Biol. 165, 566-573], in which the apical cell and its decendents normally suppress the embryogenic potential of the basal cell and its decendents during early embryo development.

Arabidopsis↗
PubMed1995

Characterization of a new myrosinase in Brassica napus.

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.

Amino Acid Sequence↗
PubMed2005

In vivo 13C NMR metabolite profiling: potential for understanding and assessing conifer seed quality.

High-resolution 13C MAS NMR spectroscopy was used to profile a range of primary and secondary metabolites in vivo in intact whole seeds of eight different conifer species native to North America, including six of the Pinaceae family and two of the Cupressaceae family. In vivo 13C NMR provided information on the total seed oil content and fatty acid composition of the major storage lipids in a non-destructive manner. In addition, a number of monoterpenes were identified in the 13C NMR spectra of conifer seeds containing oleoresin; these compounds showed marked variability in individual seeds of Pacific silver fir within the same seed lot. In imbibed conifer seeds, the 13C NMR spectra showed the presence of considerable amounts of dissolved sucrose presumed to play a protective role in the desiccation-tolerance of seeds. The free amino acids arginine and asparagine, generated as a result of storage protein mobilization, were detected in vivo during seed germination and early seedling growth. The potential for NMR to profile metabolites in a non-destructive manner in single conifer seeds and seed populations is discussed. It is a powerful tool to evaluate seed quality because of its ability to assess reserve accumulation during seed development or at seed maturity; it can also be used to monitor reserve mobilization, which is critical for seedling emergence.

Carbon Isotopes↗
PubMed1997

Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue.

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.

Amino Acid Sequence↗
PubMed1995

The Impact of Chlorophyll-Retention Mutations, d1d2 and cyt-G1, during Embryogeny in Soybean.

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.

Journal Article↗
PubMed2000

SUGAR-INDUCED SIGNAL TRANSDUCTION IN PLANTS.

Sugars have important signaling functions throughout all stages of the plant's life cycle. This review presents our current understanding of the different mechanisms of sugar sensing and sugar-induced signal transduction, including the experimental approaches used. In plants separate sensing systems are present for hexose and sucrose. Hexokinase-dependent and -independent hexose sensing systems can further be distinguished. There has been progress in understanding the signal transduction cascade by analyzing the function of the SNF1 kinase complex and the regulatory PRL1 protein. The role of sugar signaling in seed development and in seed germination is discussed, especially with respect to the various mechanisms by which sugar signaling controls gene expression. Finally, recent literature on interacting signal transduction cascades is discussed, with particular emphasis on the ethylene and ABA signal transduction pathways.

Journal Article↗