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PubMed1995

Transgenic analysis of a hybrid poplar wound-inducible promoter reveals developmental patterns of expression similar to that of storage protein genes.

The wound-inducible win3 multigene family from hybrid poplars (Populus trichocarpa x Populus deltoides) encodes proteins with structural similarities with Kunitz-type protease inhibitors (H.D. Bradshaw Jr., J.B. Hollick, T.J. Parsons, H.R.G. Clarke, M.P. Gordon [1990] Plant Mol Biol 14: 51-59), and at least one member, win3.12, is transcribed de novo in the injured and uninjured leaves of wounded trees (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). A previous study demonstrated that 1352 bp of 5' flanking DNA from the win3.12 gene confers local wound-regulated expression of the beta-glucuronidase gene in transgenic tobacco (Nicotiana tabacum cv Xanthi n.c.) (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). We extend this transgenic analysis here by examining the developmental regulation and systemic wound induction conferred by the same transgene construct in tobacco. Biochemical and histochemical surveys of beta-glucuronidase activity are described for four, independent transgenic lines. The observed spatial and temporal expression patterns coincide with dormant storage tissues and with previously described expression patterns for both seed and vegetative storage protein genes. Developmental northern blot analysis of win3 RNA levels in poplar seeds confirms that proper temporal expression of the reporter gene is maintained during tobacco seed maturation. These results demonstrate that a putative Kunitz-type protease inhibitor can be wound inducible in addition to being expressed in developing seeds.

Gene Expression Regulation, Developmental↗
PubMed1996

Characterization of the Endoproteases Appearing during Wheat Grain Development.

The pattern of endoproteolytic activities occurring during wheat (Triticum aestivum, cultivar Chinese Spring) grain development was investigated. Total endoprotease activity, assayed in solution with azocasein as a substrate, increased during the early stages of grain development to reach a maximum at 15 d postanthesis that was maintained until the grain was mature. Endoprotease activity was also assayed in gradient polyacrylamide gels co-polymerized with gelatin. The increase in endoproteolytic activity was due to the appearance of up to 18 endoproteolytic bands that were arbitrarily classified into five groups (A, B, C, D, and E). The presence of serine, aspartic, metallo, and, to a lesser extent, thiol proteases in developing wheat grains was demonstrated by the use of class-specific protease inhibitors. The appearance of the different classes of endoproteases during seed development was subject to temporal control; serine proteases were more abundant at early stages and aspartic and metallo proteases were more abundant at later stages. At intermediate stages of development (15-20 d postanthesis), most of the endoproteases were localized in the aleurone, testa, and embryo. The content of acidic thiol proteases was low in the developing starchy endosperm.

Journal Article↗
PubMed1981

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans.

The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA.

Journal Article↗
PubMed2001

Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of arabidopsis encoding highly conserved hydrophobic proteins.

The capability of most higher plants to tolerate environmental conditions strongly depends on their developmental stage. In addition, environmental factors have pleiotropic effects on many developmental processes. The interaction between plant development and environmental conditions implies that some genes must be regulated by both environmental factors and developmental cues. To understand their developmental regulation and obtain possible clues on their functions, we have isolated genomic clones for RCI2A and RCI2B, two genes from Arabidopsis ecotype Columbia (Col), whose expression is induced in response to low temperature, dehydration, salt stress, and abscisic acid. The promoters of RCI2A and RCI2B were fused to the uidA (GUS)-coding sequence and the resulting constructs used to transform Arabidopsis. GUS activity was analyzed in transgenic plants during development under both stressed and unstressed conditions. Transgenic plants with either the RCI2A or RCI2B promoter showed strong GUS expression during the first stages of seed development and germination, in vascular bundles, pollen, and most interestingly in guard cells. When transgenic plants were exposed to low temperature, dehydration, salt stress, or abscisic acid, reporter gene expression was induced in most tissues. These results indicate that RCI2A and RCI2B are regulated at transcriptional level during plant development and in response to different environmental stimuli and treatments. The potential role of RCI2A and RCI2B in plant development and stress response is discussed.

Abscisic Acid↗
PubMed1992

Transcriptional regulation of a seed-specific carrot gene, DC8.

Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to beta-glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from -170 to -51 contained ABA-responsive sequences that required a 5' upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5' upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.

Abscisic Acid↗
PubMed1998

Callose deposition is responsible for apoplastic semipermeability of the endosperm envelope of muskmelon seeds

Semipermeable cell walls or apoplastic "membranes" have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (beta-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by beta-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.

Journal Article↗
PubMed1998

Genetic analysis of female gametophyte development and function.

The female gametophyte is an absolutely essential structure for angiosperm reproduction. It produces the egg cell and central cell (which give rise to the embryo and endosperm, respectively) and mediates several reproductive processes including pollen tube guidance, fertilization, the induction of seed development, and perhaps also maternal control of embryo development. Although much has been learned about these processes at the cytological level, specific molecules mediating and controlling megagametogenesis and female gametophyte function have not been identified. A genetic approach to the identification of such molecules has been initiated in Arabidopsis and maize. Although genetic analyses are still in their infancy, mutations affecting female gametophyte function and specific steps of megagametogenesis have already been identified. Large-scale genetic screens aimed at identifying mutants affecting every step of megagametogenesis and female gametophyte function are in progress; the characterization of genes identified in these screens should go a long way toward defining the molecules that are required for female gametophyte development and function.

Arabidopsis↗
PubMed2000

Regulation of programmed cell death in maize endosperm by abscisic acid.

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2-4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.

Abscisic Acid↗
PubMed1986

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean.

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained.

Journal Article↗
PubMed2005

Requirement of aminoacyl-tRNA synthetases for gametogenesis and embryo development in Arabidopsis.

Aminoacyl-tRNA synthetases (AARSs) are required for translation in three different compartments of the plant cell: chloroplasts, mitochondria and the cytosol. Elimination of this basal function should result in lethality early in development. Phenotypes of individual mutants may vary considerably, depending on patterns of gene expression, functional redundancy, allele strength and protein localization. We describe here a reverse genetic screen of 50 insertion mutants disrupted in 21 of the 45 predicted AARSs in Arabidopsis. Our initial goal was to find additional EMB genes with a loss-of-function phenotype in the seed. Several different classes of knockouts were discovered, with defects in both gametogenesis and seed development. Three major trends were observed. Disruption of translation in chloroplasts often results in seed abortion at the transition stage of embryogenesis with minimal effects on gametophytes. Disruption of translation in mitochondria often results in ovule abortion before and immediately after fertilization. This early phenotype was frequently missed in prior screens for embryo-defective mutants. Knockout alleles of non-redundant cytosolic AARSs were in general not identified, consistent with the absolute requirement of cytosolic translation for development of male and female gametophytes. These results provide a framework for evaluating redundant functions of AARSs in Arabidopsis, a valuable data set of phenotypes resulting from multiple disruptions of a single basal process, and insights into which genes are required for both gametogenesis and embryo development and might therefore escape detection in screens for embryo-defective mutants.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

Fatty acids containing three-member carbocyclic rings are found in bacteria and plants. Bacteria synthesize cyclopropane fatty acids (CPA-FAs) only by the addition of a methylene group from S-adenosylmethionine to the cis-double bond of monoenoic phospholipid-bound fatty acids. In plants CPA-FAs are usually minor components with cyclopropene fatty acids (CPE-FAs) more abundant. Sterculia foetida seed oil contains 65-78% CPE-FAs, principally sterculic acid. To address carbocyclic fatty acid synthesis in plants, a cDNA library was constructed from developing seeds during the period of maximum oil deposition. About 0.4% of 5,300 expressed sequence tags were derived from one gene, which shared similarities to the bacterial CPA-FA synthase. However, the predicted protein is twice as large as the bacterial homolog and represents a fusion of an FAD-containing oxidase at the N terminus and a methyltransferase at the C terminus. Functional analysis of the isolated full-length cDNA was conducted in tobacco suspension cells where its expression resulted in the accumulation of up to 6.2% dihydrosterculate of total fatty acids. In addition, the dihydrosterculate was specifically labeled by [methyl-(14)C]methionine and by [(14)C]oleic acid in the transgenic tobacco cells. In in vitro assay of S. foetida seed extracts, S-adenosylmethionine served as a methylene donor for the synthesis of dihydrosterculate from oleate. Dihydrosterculate accumulated largely in phosphatidylcholine in both systems. Together, a CPA-FA synthase was identified from S. foetida, and the pathway in higher plants that produce carbocyclic fatty acids was defined as by transfer of C(1) units, most likely from S-adenosylmethionine to oleate.

Amino Acid Sequence↗
PubMed1986

Soybean seed lectin gene and flanking nonseed protein genes are developmentally regulated in transformed tobacco plants.

We introduced a 17.1-kilobase soybean DNA fragment containing the lectin gene and at least four nonseed protein genes into the tobacco genome. As in soybean plants, lectin mRNA is present in tobacco seeds, accumulates and decays during tobacco seed development, and is translated into a protein that accumulates prior to dormancy. Each soybean nonseed protein mRNA is present in tobacco leaves, roots, stems, and seeds at levels similar to that found in soybean plants. We conclude that a differentially expressed soybean gene cluster is correctly regulated in transformed tobacco plants and that sequences controlling their expression are recognized by regulatory factors present in tobacco cells.

Gene Expression Regulation↗
PubMed1996

Synthesis of small heat-shock proteins is part of the developmental program of late seed maturation.

Small heat-shock proteins (sHSPs) accumulate in plants in response to high-temperature stress. Specific sHSPs, the cytosolic class I and class II proteins, are also expressed in the absence of stress in maturing seeds of several species, and a role for these proteins in desiccation tolerance, dormancy, or germination has been hypothesized. We demonstrate that class I sHSPs are expressed during Arabidopsis seed development in a pattern similar to that previously observed in other species: they are first detected during mid-maturation, are most abundant in dry seeds, and decline rapidly during germination. Although the class I sHSP family in Arabidopsis appears to consist of four genes, expression of a single gene, Athsp 17.4, accounts for the majority of sHSPs in maturing seeds. sHSP levels were also examined in seeds of several Arabidopsis mutants with reduced sensitivity to abscisic acid inhibition, including aba1, abi1, and abi2, abi3-1, abi3-6, abi4, and abi5-1. The abi3-1 mutant has 10-fold reduced levels of sHSPs; sHSPs are undetectable in the abi3-6 mutant. All other mutants were indistinguishable from wild type. These results suggest that sHSP expression in seeds is regulated by the ABI3 response pathway and wild-type levels of sHSPs are not sufficient for seed dormancy and not necessary for desiccation tolerance. However, roles in either process cannot be ruled out. In total the data indicate that the expression of sHSPs in seeds is part of the normal developmental program of late seed maturation and the presence of sHSPs has adaptive significance for plant reproduction.

Abscisic Acid↗
PubMed1991

Distribution of myrosinase in rapeseed tissues.

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.

Journal Article↗
PubMed2005

Polymorphism and expression of isoflavone synthase genes from soybean cultivars.

Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings.

Base Sequence↗
PubMed2000

Developmentally regulated expression of two MADS-box genes, MdMADS3 and MdMADS4, in the morphogenesis of flower buds and fruits in apple.

Two MADS-box genes, MdMADS3 and MdMADS4, were isolated from the apple (Malus x domestica Borkh.) cultivar Fuji, and their spatial and temporal expression patterns were studied during morphological differentiation of the flower buds and the fruits. Both MdMADS3 and MdMADS4 showed high sequence similarities to FBP2 from petunia, TM5 from tomato, and AGL2, AGL4 from Arabidopsis. Although MdMADS3 was expressed in the inner three whorls of the floral primordium, its expression was hardly detectable in developing fruit. The second gene, MdMADS4, was ubiquitously expressed in the inflorescence meristem, floral meristem, all four floral organs, and fruit. Moreover, MdMADS4 expression was high in the vascular bundles assigned to the floral tube and the carpellary vascular bundles in fruit at early developmental stages. The MdMADS4 transcript also accumulated in embryos of the developing seeds. These results suggest that MdMADS3 and MdMADS4 are involved in different functions, and that MdMADS4 may function in the important events controlling flower and fruit development.

Amino Acid Sequence↗
PubMed1998

Expression studies of the zeaxanthin epoxidase gene in nicotiana plumbaginifolia

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

Journal Article↗
PubMed1988

Cellular localization of soybean storage protein mRNA in transformed tobacco seeds.

We transformed tobacco plants with a soybean beta-conglycinin gene that encodes the 1.7-kilobase beta-subunit mRNA. We showed that the beta-conglycinin mRNA accumulates and decays during tobacco seed development and that beta-conglycinin mRNA is undetectable in the tobacco leaf. We utilized in situ hybridization to localize beta-conglycinin mRNA within the tobacco seed. beta-Conglycinin mRNA is not detectable within the endosperm but is localized within specific embryonic cell types. The highest concentration of beta-conglycinin mRNA is found in cotyledon storage parenchyma cells. We conclude that sequences required for embryo expression, temporal control, and cell specificity are linked to the beta-conglycinin gene, and that factors regulating beta-conglycinin gene expression are compartmentalized within analogous soybean and tobacco seed regions.

Journal Article↗