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PubMed1993

The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to Brassica S-locus glycoproteins.

Non-embryogenic carrot suspension cells secrete the EP1 glycoprotein. A cDNA clone encoding EP1 was isolated and sequenced. The EP1 sequence revealed a region of homology with Brassica S-locus glycoprotein genes, an Arabidopsis S-like gene and putative S-like receptor protein kinases from maize and Arabidopsis. EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot. In developing seeds, expression was most pronounced in both the inner and outer integument epidermis.

Amino Acid Sequence↗
PubMed1994

Genetic length polymorphisms create size variation in proline-rich proteins of the cell wall.

Two genes, Prp1 and Prp2, encode proline-rich proteins that are found in different stages of developing seed coats, hypocotyls, and roots of soybeans (Glycine max (L.) Merr.). PRP1 is found in young seed coats and PRP2 is found later during seed desiccation. In some soybean varieties, both proteins are smaller as determined by immunoblotting seed coat proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that Prp1 and Prp2 genes are linked to each other by testing seed coat protein extracts from an F2 population of a cross between the cultivar Richland, which exhibits the larger PRP proteins, and Blackhawk, which has the smaller PRP proteins. The Prp1 and Prp2 genes were separated by approximately 13% recombination. Simultaneous expression of soluble PRP2 polypeptides in the maternal seed coat and underlying aleurone layer of the embryo was found. The molecular basis for the size difference between the two varieties was examined using polymerase chain reaction to isolate the Prp genes from Blackhawk, the variety that exhibited the smaller proteins. Both of the genes from Blackhawk contained length polymorphisms that result in omission of some of the repeat units (pro pro val tyr lys) from the proteins. In Prp1, there were two separate deletions in different parts of the gene, each being two tandem repeats in length. In Prp2, there was only one deletion of two tandem repeats. These deletions occur within the coding regions in a manner that conserves the reading frame. The results are the first description of genetic variation in cell wall proteins and its molecular basis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin.

Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71-75% identical. The Arabidopsis profilins bind poly-L-proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains.

Amino Acid Sequence↗
PubMed1999

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal.

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.

Amino Acid Sequence↗
PubMed2000

T-DNA insertional mutagenesis for functional genomics in rice.

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Base Sequence↗
PubMed2000

Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana.

Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta. These results indicate that AOdelta is encoded by AAO3. AOdelta produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.

Abscisic Acid↗
PubMed2003

Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family.

A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.

Abscisic Acid↗
PubMed2000

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.

Animals↗
PubMed2003

Effect of acute drought stress and time of harvest on phytochemistry and dry weight of St. John's wort leaves and flowers.

The phytochemistry and dry weight of cultivated St. John's wort are significantly influenced by acute drought stress and time of harvest. In this study, plants subjected to brief drought stress during both flower and seed development periods exhibited increased concentrations in 8 of the 10 phytochemicals examined in this study, including hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, and quercetin. Increases ranged from 5% to 36% (hyperoside and rutin, respectively). Conversely, the concentrations of hyperforin and adhyperforin in flowers were decreased by an average of 10% in drought-stressed plants as compared to well-watered control plants. Acute drought stress decreased flower dry weight significantly during both drydown periods, although vegetative parameters (height, leaf dry weight and stem dry weight) were not adversely affected. While acute drought stress significantly altered the chemical yield in the leaves and flowers (phytochemical content x harvested dry weight), the time of harvest was the predominant factor determining phytochemical concentration in the organs of H. perforatum.

Adaptation, Physiological↗
PubMed2006

Formation of two methylenedioxy bridges by a Sesamum CYP81Q protein yielding a furofuran lignan, (+)-sesamin.

(+)-Sesamin, a furofuran class lignan, is widespread in vascular plants and represented by Sesamum spp. (+)-Sesamin has been of rapidly growing interest because of its beneficial biological effects in mammals, but its biosynthesis and physiological roles in plants remain to be clarified. It is speculated to be synthesized from (+)-pinoresinol by means of (+)-piperitol by formation of two methylenedioxy bridges mediated by two distinct Sesamum indicum cytochrome P450 (SiP450) proteins. Here, we report an SiP450, CYP81Q1, that alone catalyzes (+)-sesamin biosynthesis from (+)-pinoresinol by means of (+)-piperitol by forming two methylenedioxy bridges. The CYP81Q1 gene expression profile was temporally consistent with the accumulation pattern of (+)-sesamin during seed development. The CYP81Q1-GFP chimera protein was colocalized with an endoplasmic reticulum (ER)-targeting chimera protein, indicating that (+)-sesamin biosynthesis occurs on the ER cytoplasmic surface. Moreover, we isolated two CYP81Q1 homologs from other Sesamum spp. Sesamum radiatum CYP81Q2 showed dual (+)-piperitol/(+)-sesamin synthetic activity. CYP81Q2, as well as CYP81Q1, therefore, corresponds to a (+)-piperitol/(+)-sesamin synthase in lignan biosynthesis. In contrast, Sesamum alatum CYP81Q3 showed no activity, in accord with (+)-sesamin being deficient in S. alatum. Our findings not only provide insight into lignan biosynthesis but also unravel a unique mode of cytochrome P450 action.

Amino Acid Sequence↗
PubMed1985

Developmentally regulated expression of the bean beta-phaseolin gene in tobacco seed.

Recombinant phage lambda177.4 contains a gene for beta phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-BamHI fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.

Journal Article↗
PubMed1989

Heat shock protein hsp70 cognate gene expression in vegetative and reproductive organs of Lycopersicon esculentum.

We have detected hsc70 gene expression (heat shock protein hsp70 cognate) during vegetative growth and reproductive development in tomato (Lycopersicon esculentum). Using RNA from a tomato hsc70 cDNA as a probe in in situ hybridizations, we have determined expression patterns of hsc70 in nonstressed tomato roots, stems, leaves, flowers, and developing fruits. We have localized high levels of hsc70 transcript to the vascular system of the ovary, dividing cells of the lateral root tips, and the inner integument of developing seeds. We also see expression in the transmitting tissue, in immature anthers, and in embryos. We cannot detect expression in mature pollen, xylem, or ovules. These data indicate that the expression of at least some tomato hsp70 family members is developmentally regulated.

Journal Article↗
PubMed1994

Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase.

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.

Base Sequence↗
PubMed1996

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.

Alleles↗
PubMed1997

The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis.

APETALA2 (AP2) plays an important role in the control of Arabidopsis flower and seed development and encodes a putative transcription factor that is distinguished by a novel DNA binding motif referred to as the AP2 domain. In this study we show that the AP2 domain containing or RAP2 (related to AP2) family of proteins is encoded by a minimum of 12 genes in Arabidopsis. The RAP2 genes encode two classes of proteins, AP2-like and EREBP-like, that are defined by the number of AP2 domains in each polypeptide as well as by two sequence motifs referred to as the YRG and RAYD elements that are located within each AP2 domain. RAP2 genes are differentially expressed in flower, leaf, inflorescence stem, and root. Moreover, the expression of at least three RAP2 genes in vegetative tissues are controlled by AP2. Thus, unlike other floral homeotic genes, AP2 is active during both reproductive and vegetative development.

Amino Acid Sequence↗
PubMed1997

Mendel's dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins.

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3beta-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3beta-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le "mutation" and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 microM and 13 microM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3beta-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3beta-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

Alleles↗
PubMed1998

The trihelix DNA-binding motif in higher plants is not restricted to the transcription factors GT-1 and GT-2.

GT-2 is a plant transcriptional activator that contains two separate, but similar, trihelix DNA-binding domains. GT-1 is similar to GT-2, although it contains only one of such domains. cDNAs that encode GT-2 were isolated from rice (OS-GT2) and Arabidopsis (AT-GT2). Evidence is presented for the existence of an Arabidopsis gene family that is structurally related to AT-GT2. Two members of this GT2-like family, AT-GTL1 and AT-GTL2, have been isolated and characterized. Their sequences suggest that they evolved by a recent gene duplication event. Both AT-GT2 and AT-GTL genes contain an intron in the amino-terminal trihelix motif, indicating that this DNA-binding domain resulted from exon shuffling. RNA gel blot analysis using AT-GTL1 as a probe revealed four transcripts in the aerial part of the plant. All mRNA levels were significantly higher in siliques, suggesting that this gene family may function in fruit and/or seed development. To date, DNA-binding proteins characterized by the trihelix motif have been described only in plants, and may therefore be involved in plant-specific processes. Our results show that in Arabidopsis thaliana, the trihelix motif is not restricted to the GT-1 and GT-2 DNA-binding proteins.

Amino Acid Sequence↗
PubMed1999

Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos. alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

Amino Acid Sequence↗