Showing papers in "The Plant Cell in 1990"

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.

Abstract: We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.

Early flower development in Arabidopsis.

Abstract: The early development of the flower of Arabidopsis thaliana is described from initiation until the opening of the bud The morphogenesis, growth rate, and surface structure of floral organs were recorded in detail using scanning electron microscopy Flower development has been divided into 12 stages using a series of landmark events Stage 1 begins with the initiation of a floral buttress on the flank of the apical meristem Stage 2 commences when the flower primordium becomes separate from the meristem Sepal primordia then arise (stage 3) and grow to overlie the primordium (stage 4) Petal and stamen primordia appear next (stage 5) and are soon enclosed by the sepals (stage 6) During stage 6, petal primordia grow slowly, whereas stamen primordia enlarge more rapidly Stage 7 begins when the medial stamens become stalked These soon develop locules (stage 8) A long stage 9 then commences with the petal primordia becoming stalked During this stage all organs lengthen rapidly This includes the gynoecium, which commences growth as an open-ended tube during stage 6 When the petals reach the length of the lateral stamens, stage 10 begins Stigmatic papillae appear soon after (stage 11), and the petals rapidly reach the height of the medial stamens (stage 12) This final stage ends when the 1-millimeter-long bud opens Under our growing conditions 19 buds were initiated per day on average, and they took 1325 days to progress through the 12 stages from initiation until opening

Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

Abstract: To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Abstract: Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

Abstract: A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Gene expression in response to abscisic acid and osmotic stress.

Abstract: Abscisic acid (ABA) was discovered in the 1950s to be a phytohormone affecting leaf abscision and bud dormancy. It was soon characterized as a sesquiterpene derived from mevalonate although certain steps of its biosynthesis in plants are still unknown (Li and Walton, 1987; Zeevaart and Creelman, 1988). Continuing work on ABA has shown that it mediates various developmental and physiological processes that affect the agronomic performance of crop plants (Austin et al., 1982; Ramagopal, 1987). These proc? esses include embryo maturation and germination as well as the response of vegetative tissues to osmotic stress (Singh et al., 1987; Zeevaart and Creelman, 1988). ABA levels increase in tissues subjected to osmotic stress by desiccation, salt, or cold (Henson, 1984; Mohapatra et al., 1988). Under these conditions, specific genes are ex? pressed that can also be induced in unstressed tissues by the application of exogenous ABA (Singh et al., 1987; Gomez et al., 1988; Mundy and Chua, 1988). Some of these genes are also expressed during the normal embryogenic program when seeds desiccate and embryos be? come dormant (Dure et al., 1981). Although different sets of ABA-responsive genes exhibit different patterns of de? velopmental and tissue-specific expression, some of them appear to be part of a general reaction to osmotic stress. This system is a normal part of the embryogenic program but is inducible in vegetative tissues at other times in the plant life cycle. Several ABA-responsive genes have now been isolated (Baker et al., 1988; Gomez et al., 1988; Marcotte et al., 1988; Mundy and Chua, 1988; Vilardell et al., 1990; Yamaguchi-Shinozaki et al., 1990). A major goal of the research discussed below is to understand the role these genes play in osmotic stress and desiccation tolerance.

Isolation of an efficient actin promoter for use in rice transformation

Abstract: We have characterized the 5' region of the rice actin 1 gene (Act1) and show that it is an efficient promoter for regulating the constitutive expression of a foreign gene in transgenic rice. By constructing plasmids with 5' regions from the rice Act1 gene fused to the coding sequence of a gene encoding bacterial beta-glucuronidase, we demonstrate that a region 1.3 kilobases upstream of the Act1 translation initiation codon contains all of the 5'-regulatory elements necessary for high-level beta-glucuronidase (GUS) expression in transient assays of transformed rice protoplasts. The rice Act1 primary transcript has a noncoding exon separated by a 5' intron from the first coding exon. Fusions that lack this Act1 intron showed no detectable GUS activity in transient assays of transformed rice protoplasts. Deletion analysis of the Act1 5' intron suggests that the intron-mediated stimulation of GUS expression is associated, in part, with an in vivo requirement for efficient intron splicing.

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

Abstract: We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Metabolic repression of transcription in higher plants.

Abstract: Using freshly isolated maize mesophyll protoplasts and a transient expression method, I showed that the transcriptional activity of seven maize photosynthetic gene promoters is specifically and coordinately repressed by the photosynthetic end products sucrose and glucose and by the exogenous carbon source acetate. Analysis of deleted, mutated, and hybrid promoters showed that sugars and acetate inhibit the activity of distinct positive upstream regulatory elements without a common consensus. The metabolic repression of photosynthetic genes overrides other forms of regulation, e.g., light, tissue type, and developmental stage. Repression by sugars and repression by acetate are mediated by different mechanisms. The identification of conditions that avoid sugar repression overcomes a major obstacle to the study of photosynthetic gene regulation in higher plants.

Arabidopsis is susceptible to infection by a downy mildew fungus.

Abstract: A population of Arabidopsis thaliana growing locally in a suburb of Zurich called Weiningen was observed to be infected with downy mildew. Plants were collected and the progress of infection was investigated in artificial inoculations in the laboratory. The plants proved to be highly susceptible, and pronounced intercellular mycelial growth, haustoria formation, conidiophore production, and sporulation of the causal organism Peronospora parasitica were all observed. The formation of oogonia, antheridia, and oospores also occurred. In contrast, Arabidopsis strain RLD was resistant to infection and none of the above structures was formed. The fungus was localized very soon after penetration of RLD leaf cells, which responded with a typical hypersensitive reaction. The differential interaction of an isolate of P. parasitica with two strains of Arabidopsis opens up the possibility of cloning resistance determinants from a host that is very amenable to genetic and molecular analysis.

Growth and development of the axr1 mutants of Arabidopsis.

Abstract: We have recovered eight new auxin-resistant lines of Arabidopsis that carry mutations in the AXR1 gene. These eight lines, together with the 12 lines described in a previous report, define at least five different axr1 alleles. All of the mutant lines have a similar phenotype. Defects include decreases in plant height, root gravitropism, hypocotyl elongation, and fertility. Mutant line axr1-3 is less resistant to auxin than the other mutant lines and has less severe morphological abnormalities. This correlation suggests that the morphological defects are a consequence of a defect in auxin action. To determine whether the altered morphology of mutant plants is associated with changes in cell size or tissue organization, tissue sections were examined using scanning electron microscopy. No clear differences in cell size were observed between wild-type and mutant tissues. However, the vascular bundles of mutant stems were found to be less well differentiated than those in wild-type stems. The auxin sensitivity of rosette-stage plants was determined by spraying plants with auxin solutions. Mutant rosettes were found to be significantly less sensitive to exogenously applied auxin than wild-type rosettes, indicating that the AXR1 gene functions in aerial portions of the plant. Our studies suggest that the AXR1 gene is required for auxin action in most, if not all, tissues of the plant and plays an important role in plant development. Linkage studies indicate that the gene is located on chromosome 1 approximately 2 centiMorgans from the closest restriction fragment length polymorphism.

Function of the apetala-1 gene during Arabidopsis floral development.

Abstract: We have characterized the floral phenotypes produced by the recessive homeotic apetala 1-1 (ap1-1) mutation in Arabidopsis. Plants homozygous for this mutation display a homeotic conversion of sepsis into brachts and the concomitant formation of floral buds in the axil of each transformed sepal. In addition, these flowers lack petals. We show that the loss of petal phenotype is due to the failure of petal primordia to be initiated. We have also constructed double mutant combinations with ap1 and other mutations affecting floral development. Based on these results, we suggest that the AP1 and the apetala 2 (AP2) genes may encode similar functions that are required to define the pattern of where floral organs arise, as well as for determinate development of the floral meristem. We propose that the AP1 and AP2 gene products act in concert with the product of the agamous (AG) locus to establish a determinate floral meristem, whereas other homeotic gene products are required for cells to differentiate correctly according to their position. These results extend the proposed role of the homeotic genes in floral development and suggest new models for the establishment of floral pattern.

Molecular light switches for plant genes.

Abstract: Light is essential for normal plant growth and development not only as a source of energy but also as a stimulus that regulates numerous developmental and metabolic proc? esses. The plant's responses are varied and complex and dependent upon the quality and quantity of ambient light. The initial requirement for light is as a signal for germination in many plant species. After germination in complete dark? ness, seedlings have a morphology distinct from lightgrown ones and do not express light-inducible genes. Upon illumination of these etiolated seedlings, modifications in the transcription of light-responsive genes occur and rapid light-induced morphological changes ensue. Adaptation of plants in continuous darkness for 2 to 3 days does not cause dramatic morphological changes but results in alter? ations of specific transcript levels (for recent reviews, see Ellis, 1986; Kendrick and Kronenberg, 1986; Cuozzo et al., 1987; Kuhlemeier etal., 1987b; Silverthorne and Tobin, 1987; Jenkins, 1988; Nagy et al., 1988). The most extensively studied light-responsive genes are those encoding the small subunit of ribulose-1,5-bisphos? phate carboxylase-oxygenase (rbcS) and the chlorophyll a/?>-binding proteins (cab) (see Tobin and Silverthorne, 1985; Manzara and Gruissem, 1988; Dean et al., 1989c). In several plant species an increase in the transcript levels from these genes occurs in etiolated seedlings and darkadapted plants in response to light. This increase is me? diated by the photoreceptor phytochrome and is regulated at the transcriptional level (Gallagher and Ellis, 1982; Sil? verthorne and Tobin, 1984; Berry-Lowe and Meagher, 1985; Mosinger et al., 1985). Phytochrome is the best characterized of the three known photoreceptors. The other two, cryptochrome and the UV-B photoreceptor, mediate their effects in response to blue and UV light, respectively (see Kendrick and Kronenberg, 1986). The expression of many light-responsive genes is modulated by more than one wavelength of light (see Tobin and Silverthorne, 1984; Ellis, 1986; Kuhlemeier et al., 1987b). The observation that different light responses are mediated through distinct photoreceptors raises the question of whether genes that respond to more than one wavelength do so through distinct c/s-acting elements or whether the signal transduction pathways converge to act upon the same regulatory sequence. Analyses of the kinetics of light-responsive gene induc? tion show that the rate of mRNA accumulation is variable among genes and can be dependent on the developmental state of the plant (Gallagher et al., 1985; Fluhr and Chua, 1986). This variation may be due to a requirement for distinct regulatory factors or because the genes have different thresholds for a specific regulator. Several genes are down-regulated by light, specifically those encoding phytochrome (Lissemore and Quail, 1988; Kay et al., 1989), NADPH-protochlorophyllide reductase (Batschauer and Apel, 1984; Darrah et al., 1990), and asparagine synthetase (Tsai and Coruzzi, 1990). For each of these genes, the photoresponse is mediated by phyto? chrome. The ability of one photoreceptor to mediate op? posite patterns of expression implies that there is a branch point in the signal transduction pathway leading to these different responses. Studies of many light-regulated genes from different species demonstrate that DNA elements responsible for light-responsive expression are located within 5' upstream sequences (see Kuhlemeier et al., 1987b; Silverthorne and Tobin, 1987; Jenkins, 1988; Benfey and Chua, 1989; Dean et al., 1989a; Stockhaus et al., 1989). However, there is evidence that other regions of the gene can mediate changes in transcript abundance in response to light. For example, in the case of a pea gene encoding ferredoxin, sequences within the transcribed region modulate mRNA levels by affecting transcript stability (Elliot et al., 1989a). In addition, nuclear run-on experiments with petunia rbcS show that both upstream and downstream sequences play a role in the transcriptional regulation of these genes (Dean et al., 1989b). In contrast, downstream sequences of pea rbcS do not affect steady-state transcript abundance (Kuhlemeier et al., 1988b). These differences may reflect subtle variations in the mechanisms that operate to regu? late rbcS expression in different plant species. 1 Current address: Department of Plant Molecular Biology, Leiden University, Clusius Laboratory, Lassenaarseweg 64, 2333 AL Leiden, The Netherlands. 2 To whom correspondence should be addressed.

Genetic Control of Root Hair Development in Arabidopsis thaliana.

Abstract: Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Abstract: Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

Nuclear transport of plant potyviral proteins.

Abstract: We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the beta-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-Nlb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

Abstract: We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Abstract: Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

Abstract: Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis.

Abstract: Phenylalanine ammonia-lyase (PAL) is encoded by a small family of genes in Arabidopsis. We cloned and partially characterized one of these genes, PAL1. The deduced amino acid sequence is highly similar to PAL from bean, parsley, and rice. The promoter contains sequence elements homologous to two putative regulatory elements conserved among several phenylpropanoid genes. The regulation of the PAL1 gene was examined by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing PAL1-GUS gene fusions. The PAL1 promoter was activated early in seedling development and in adult plants was strongly expressed in the vascular tissues of roots and leaves, but was not active in the root tip or the shoot apical meristem. In flowers, expression was observed in sepals, anthers, and carpels, but not in petals. Transcripts encoded by the endogenous PAL genes and GUS transcripts from the PAL1-GUS gene fusion were induced by wounding, HgCl2-stress, and light. Analysis of the regulatory properties of 5' deleted promoters showed that the proximal region of the promoter to -290 was sufficient to establish the full tissue-specific pattern of expression and that the proximal region to -540 was responsive to environmental stimuli. Negative and positive elements were located between -1816 and -823 and between -823 and -290, respectively.

Protein secretion in plant cells can occur via a default pathway.

Abstract: To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.

Transient Gene Expression in Intact and Organized Rice Tissues.

Abstract: Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a [beta]-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light-harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.

Sequential induction of Nodulin gene expression in the developing Pea Nodule

Abstract: A set of cDNA clones have been characterized that represent early nodulin mRNAs from pea root nodules. By RNA transfer blot analyses, the different early nodulin mRNAs were found to vary in time course of appearance during the development of the indeterminate pea root nodule. In situ hybridization studies demonstrated that the transcripts were located in different zones, representing subsequent steps in development of the central tissue of the root nodule. ENOD12 transcripts were present in every cell of the invasion zone, whereas ENOD5, ENOD3, and ENOD14 transcripts were restricted to the infected cells in successive but partially overlapping zones of the central tissue. We conclude that the corresponding nodulin genes are expressed at subsequent developmental stages. The amino acid sequence derived from the nucleotide sequences of the cDNAs, in combination with the localization data, showed that ENOD5 is an arabinogalactan-like protein involved in the infection process, whereas ENOD3 and ENOD14 have a cysteine cluster suggesting that these are metal-binding proteins. Furthermore, we showed that there is a clear difference in the way Rhizobium induced the infection-related early nodulin genes ENOD5 and ENOD12. A factor acting over a long distance induced the ENOD12 gene, whereas a factor acting over a short distance activated the ENOD5 gene.

Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize.

Abstract: A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.

Expression of Two Soybean Vegetative Storage Protein Genes during Development and in Response to Water Deficit, Wounding, and Jasmonic Acid

Abstract: The expression of vspA and vspB genes encoding soybean vegetative storage proteins was studied during seedling development and in response to water deficit, tissue wounding, and jasmonic acid treatment. vspA and vspB encode VSP-a and VSP-8, 28-kilodalton and 31-kilodalton vacuole-localized polypeptides that are 80% homologous. vspA and vsp6 mRNAs could be distinguished on RNA blots using 3’-end probes. vspA mRNA was threefold to sevenfold more abundant than vsp6 mRNA in leaves, about equal expression was observed in stems, and vsp6 mRNA exceeded vspA in roots. Transcripts were not detected in dry seeds but appeared in intact or excised seedling axes between 12 hr and 24 hr after initiation of imbibition. Both transcripts were highly abundant in the meristematic region of seedling stems and in developing leaves but were rare in mature stems, leaves, and roots. In situ localization showed that vsp transcripts were found throughout the hypocotyl hook but were concentrated in cells associated with the epidermis and vascular bundles. Water deficit caused increased vsp mRNA levels in leaves and stems, which suggests that inhibition of growth necessitates temporary storage of amino acids. Wounding induced primarily vsp6 mRNA in etiolated seedlings, whereas both vspA and vsp6 mRNA levels increased in wounded leaves. Jasmonic acid and methyl jasmonate were potent inducers of vsp gene expression in cell cultures, developing axes, leaves, and roots. We hypothesize that jasmonic acid levels modulate vsp mRNA abundance in vivo.

Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions

Abstract: Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym [Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D. (1989). In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems. (Berlin: Springer-Verlag), pp. 401-407]. To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene. Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them. Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline. None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism. trc fusions exhibiting a low basal and a high induced [beta]-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions. Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association.

Novel Regulation of Vegetative Storage Protein Genes.

Abstract: Until recently, storage protein research has been largely restricted to the seed-specific reserve proteins, which provide nutrients to seedlings during their early growth. Their abundance, and the potential for improving seed nutritional quality, were early incentives to investigate seed storage proteins, and they continue to be intensively studied as a plant model for tissue-specific and developmentally regulated genes. Recent research has shown that non-seed tissues also synthesize proteins that are distinct from the seed reserves but have the characteristics of storage proteins. That is, they are expressed abundantly in specific cells, are localized in vacuoles (the progenitors of protein bodies in seeds), and are degraded at a later time for the nutritional needs of other organs. It is perhaps not surprising that vegetative tissues contain reserve proteins since plants assimilate, translocate, and reutilize nutrients extensively throughout their life cycle (for reviews, see Pate, 1980; Simpson, 1986). This review focuses on a soybean vegetative storage protein (VSP) that is particularly important for the temporary storage of nitrogen. Unlike the seed proteins, whose massive accumulation comes at the end of a developmental stage, the VSP genes are highly modulated throughout development according to the immediate need to store surplus nitrogen or amino acids. They are particularly intriguing and attractive for further studies because their expression is enhanced by severa1 different stimuli, including seed pod removal, high nitrogen nutrition, and wounding. Jasmonic acid, a nove1 plant compound and a possible endogenous gene regulator, also induces VSP gene expression. As with their seed counterparts, the VSP genes may be important targets for increasing plant productivity or nutritional quality through genetic engineering.

Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant.

Abstract: Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development.

Peroxidase-Induced Wilting in Transgenic Tobacco Plants.

Abstract: Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development, or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H2O2 or O2 to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, we have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. We were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated at the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increased water loss through the leaf surface or stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.

An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF).

Abstract: The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.