Showing papers in "The Plant Cell in 1989"

Genes directing flower development in Arabidopsis.

Abstract: We describe the effects of four recessive homeotic mutations that specifically disrupt the development of flowers in Arabidopsis thaliana. Each of the recessive mutations affects the outcome of organ development, but not the location of organ primordia. Homeotic transformations observed are as follows. In agamous-1, stamens to petals; in apetala2-1, sepals to leaves and petals to staminoid petals; in apetala3-1, petals to sepals and stamens to carpels; in pistillata-1, petals to sepals. In addition, two of these mutations (ap2-1 and pi-1) result in loss of organs, and ag-1 causes the cells that would ordinarily form the gynoecium to differentiate as a flower. Two of the mutations are temperature-sensitive. Temperature shift experiments indicate that the wild-type AP2 gene product acts at the time of primordium initiation; the AP3 product is active later. It seems that the wild-type alleles of these four genes allow cells to determine their place in the developing flower and thus to differentiate appropriately. We propose that these genes may be involved in setting up or responding to concentric, overlapping fields within the flower primordium.

Abscisic acid-responsive sequences from the em gene of wheat.

Abstract: We demonstrate that a chimeric gene containing the beta-glucuronidase (GUS) reporter gene linked to a 646-base pair 5' fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to -103) fused 5' in either orientation to a truncated cauliflower mosaic virus promoter (35S) increases GUS activity threefold in the presence of ABA. Insertion of the Em 5'-untranslated region (+6 to +86) between the 35S promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5' region: an ABA response element from -152 to -103 and an element between +6 and +86 that quantitatively increases the ABA response.

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Abstract: Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

Abstract: Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.

Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences.

Abstract: Genetic studies in maize have identified several regulatory genes that control the tissue-specific synthesis of the purple anthocyanin pigments during development. Two such genes, R and B, exhibit extensive allelic diversity with respect to the tissue specificity and developmental timing of anthocyanin synthesis. Previous genetic studies demonstrated that certain B alleles can substitute for R function, and in these cases only one functional allele at either locus is required for pigment synthesis in the aleurone. In addition, biochemical studies have shown that both genes act on the same biosynthetic pathway, suggesting that the genes are functionally duplicate. In this report we describe DNA hybridization experiments that demonstrate that the functionally duplicate nature of B and R is reflected in DNA sequence similarity between the two genes. We took advantage of this homology and used the R genomic sequences to clone B. Two different strategies were pursued and two genomic clones isolated, a 2.5-kilobase BgIII fragment linked to the b allele in W23 inbred stocks and a 1.0-kilobase HindIII fragment linked to the B allele in CM37 stocks. Examination of several independent transposable element insertion mutations in B and revertant derivatives demonstrated that our clones recognize the functional B gene. Genomic clones representing the entire B-Peru allele were isolated, and a detailed restriction map was prepared. Using these clones we have identified a 2.2-kilobase mRNA in husks from plants containing either B-I or B-Peru alleles, but no B mRNA was detected in plants containing a b allele. The transcript is at least 100 times more abundant in strongly pigmented B-I husks than in weakly pigmented B-Peru husk tissue. Expression of functional B alleles in husk tissue correlates with the coordinate increase in mRNA levels of two structural genes of the pathway, A1 and Bz1, consistent with the postulated role of B as a regulatory gene.

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Abstract: Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

Characterization of the glycinin gene family in soybean.

Abstract: We characterized the structure, organization, and expression of genes that encode the soybean glycinins, a family of storage proteins synthesized exclusively in seeds during embryogenesis. Five genes encode the predominant glycinin subunits found in soybeans, and they have each been cloned, sequenced, and compared. The five genes have diverged into two subfamilies that are designated as Group-I and Group-II glycinin genes. Each glycinin gene contains four exons and three introns like genes that encode related proteins in other legumes. Two other genes have been identified and designated as "glycinin-related" because they hybridize weakly with the five glycinin genes. Although not yet characterized, glycinin-related genes could encode other glycinin subunit families whose members accumulate in minor amounts in seeds. The three Group-I glycinin genes are organized into two chromosomal domains, each about 45 kilobase pairs in length. The two domains have a high degree of homology, and contain at least five genes each that are expressed either in embryos or in mature plant leaves. Gel blot studies with embryo mRNA, as well as transcription studies with 32P-RNA synthesized in vitro from purified embryo nuclei, indicate that glycinin and glycinin-related genes become transcriptionally activated in a coordinated fashion early in embryogenesis, and are repressed coordinately late in seed development. In addition to transcriptional control processes, posttranscriptional events also are involved in regulating glycinin and glycinin-related mRNA levels during embryogenesis.

Changes in the zein composition of protein bodies during maize endosperm development.

Abstract: Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants.

Abstract: The 35S promoter is a major promoter of the cauliflower mosaic virus that infects crucifers. This promoter is still active when excised from cauliflower mosaic virus and integrated into the nuclear genome of transgenic tobacco. Previous work has shown that the -343 to -46 upstream fragment is responsible for the majority of the 35S promoter strength (Odell, J.T., Nagy, F., and Chua, N.-H. [1985]. Nature 313, 810-812). Here we show by 5', 3', and internal deletions that this upstream fragment can be subdivided into three functional regions, -343 to -208, -208 to -90, and -90 to -46. The first two regions can potentiate transcriptional activity when tested with the appropriate 35S promoter sequence. In contrast, the -90 to -46 region by itself has little activity but it plays an accessory role by increasing transcriptional activity of the two distal regions. Finally, we show that monomers and multimers of a 35S fragment (-209 to -46) can act as enhancers to potentiate transcription from a heterologous promoter.

Molecular Analysis of viviparous-1: An Abscisic Acid-Insensitive Mutant of Maize.

Abstract: The viviparous-1 (vp1) gene in maize controls multiple developmental responses associated with the maturation phase of seed formation. Most notably, mutant embryos have reduced sensitivity to the hormone abscisic acid, resulting in precocious germination, and blocked anthocyanin synthesis in aleurone and embryo tissues. The Vp1 locus was cloned by transposon tagging, using the Robertson's Mutator element present in the vp1-mum1 mutant allele. Detection of DNA rearrangements in several spontaneous and transposable element-induced mutant vp1 alleles, including a partial deletion of the locus, confirmed the identity of the clone. The Vp1 gene encodes a 2500-nucleotide mRNA that is expressed specifically in embryo and endosperm tissues of the developing seed. This transcript is absent in seed tissues of vp1 mutant stocks. Expression of C1, a regulatory gene for the anthocyanin pathway, is selectively blocked at the mRNA level in vp1 mutant seed tissues, indicating the Vp1 may control the anthocyanin pathway by regulating C1. We suggest that the Vp1 gene product functions to potentiate multiple signal transduction pathways in specific seed tissues.

Pathogenesis-related proteins are developmentally regulated in tobacco flowers.

Abstract: The accumulation of pathogenesis-related proteins (PR) in tobacco leaves has been casually related to pathogen and specific physiological stresses. The known enzymatic function of some of these proteins is potentially antimicrobial. By using antibodies specific to three classes of pathogenesis-related proteins, we examined tobacco plants during their normal growth. The pathogenesis-related proteins accumulated during the normal development of the tobacco flower. The PR-1 class of proteins (biological function unknown) is located in sepal tissue. PR-P, Q polypeptides are endochitinases and are present in pedicels, sepals, anthers, and ovaries. A glycoprotein serologically related to the PR-2,N,O class is a (1,3)-beta-glucanase and is present in pistils. Differential appearance during flower development, in situ localization, and post-translational processing of floral pathogenesis-related proteins point to a hitherto unsuspected function these classes of pathogenesis-related proteins play in the normal process of flowering and reproductive physiology.

Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene.

Abstract: A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

Abstract: We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.

AP2 Gene Determines the Identity of Perianth Organs in Flowers of Arabidopsis thaliana.

Abstract: We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.

Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

Abstract: S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants.

Abstract: We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A thaliana hy mutants Among six complementation groups that define the HY phenotype in A thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A thaliana seedlings When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (eg, gene expression) associated with chloroplast development

The Male Gametophyte of Flowering Plants.

Abstract: Angiosperm plants produce flowers, very beautiful and intricate structures, within which their reproductive devel? opment takes place. In flowering plants, as in other groups of plants, a diploid, spore-producing generation (sporo? phyte) altemates with a haploid, gamete-producing gen? eration (gametophyte). Unlike some of the evolutionarily more primitive plants, the male and female gametophytes of angiosperms are reduced to microscopic structures that are dependent on the tissues of the sporophyte for their development. The flower contains specialized structures, the anthers and the pistil or gynoecium, in which the male and female gametophytes, respectively, are formed. The functions of the gametophytes are the production of the sperm cells and the female cells, and their union in fertilization. In flowering plants, the pollen grain is the male gametophyte and the embryo sac is the female gametophyte. The male gametophyte completes its early development within the anther. The sequential stages of pollen devel? opment are shown in Figure 1. Microsporocytes or pollen mother cells (Figures 1A and 1K) are produced in the sporogenous tissue within the anther. The two divisions of meiosis transform these cells into haploid microspores, each pollen mother cell producing first a dyad (Figures 1B and 1L) and, after the second meiotic division, a tetrad of microspores (Figures 1C and 1M). The tetrad and each microspore within the tetrad are surrounded by a callose (1,3-?-glucan) wall (Figure 1M). Upon release from the tetrad, the microspores increase rapidly in volume and undergo a change in shape (Figures 1D, 1E, 1N, and 10). This is followed by a period of slower growth until the maximum volume of the pollen grains is reached before anthesis. Following release of microspores from the te? trads, there is an extended interphase period that terminates with a very unequal division of the microspore (mi? crospore mitosis), forming a vegetative cell and a generative cell, both of which are included within the confines of the cell wall of the original microspore. The vegetative cell constitutes the bulk of the young pollen grain, while the generative cell, which inherits a very small amount of the microspore cytoplasm, lies within the vegetative cell (Fig? ures 1F and 1G). In several plants, such as corn, the generative cell undergoes a mitotic division within the pollen grain, forming two sperm cells (Figures 1H and 1R). In most pollens, however, the generative cell completes its division during the growth of the pollen tube in the style. At ma urity, the male gametophyte consists of three cells, the vegetative cell and the two sperm cells, which lie within the cytoplasm of the vegetative cell (Figures 11 and 1S). For a short while following anthesis (the rupture of the anther and release of pollen), the mature pollen grain exists as a free organism until it is transported by wind, insects, or other agents to the stigma of an appropriate pistil. It then begins another phase of its life and development. Each pollen grain germinates by the extrusion of a tube through a germ pore in the pollen wall (Figures 1J and 1T). The tube then grows down into the style and the vegetative nucleus, and, depending on the plant species, the gener? ative cell or sperm cells move out of the pollen grain and i to the tube. Germination and pollen tube growth are relatively rapid events in most plants, the period from pollination to fertilization ranging from 1 hr to around 48 hr. The rate of pollen tube growth varies in different plant species; extremely rapid rates as high as 35 mm/hr have been reported for some plants. The pollen tube grows through the transmitting tissue of the style, enters the micropyle of the ovule, and reaches the embryo sac. It penetrates one of the synergids of the embryo sac (nor? mally the one that has begun to degenerate), the tube rup ures, and the sperm cells together with some of the other tube contents are discharged into the synergid. The two sperm move by a presently unknown mechanism, one fusing with the egg cell to form the diploid zygote. The other sperm fuses with the normally diploid central cell, giving rise to the primary endosperm nucleus. This com? pletes the process of double fertilization. A major recent advance is the observation that the two sperm cells in a pollen grain are often morphologically different (Russell,

Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.

Abstract: We have characterized a soybean gene cluster that encodes a group of auxin-regulated RNAs (small auxin up RNAs). DNA sequencing of a portion of the locus reveals five homologous genes, spaced at intervals of about 1.25 kilobases and transcribed in alternate directions. At least three of the genes are transcriptionally regulated by auxin. An increase in the rate of transcription is detected 10 min after application of auxin to soybean elongating hypocotyl sections. Each of the genes contains an open reading frame that could encode a protein of 9 kilodaltons to 10.5 kilodaltons. Sequence comparisons among the five genes reveal several areas of high homology. Two regions of high homology begin about 250 base pairs upstream of the open reading frames and two regions of homology have been identified in sequences downstream of the open reading frames. One of the latter sequences occurs in the 39-untranslated region of the RNAs. The other occurs far downstream, 618 base pairs to 741 base pairs from the stop codon. Conservation of these sequences among the five different genes suggests that they may be important for the regulation of expression of the genes.

Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.

Abstract: In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.

Restriction Fragment Length Polymorphism Linkage Map of Arabidopsis thaliana.

Abstract: We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.

Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata.

Abstract: Three alleles of the self-incompatibility gene of Nicotiana alata have been cloned and sequenced. A comparison of the sequences shows a surprisingly low level of homology (56%) and the presence of defined regions of homology and variability. The homologous regions include the N-terminal sequence, most of the cysteine residues and glycosylation sites, as well as other blocks throughout the sequence. We interpret these conserved regions as "framework" and nonconserved regions as "hypervariable," following the terminology used to describe analogous regions in the IgG supergene family. The low level of overall homology forms the basis of a general method for isolating S-allele-specific cDNAs. Allele-specific antibodies can be generated using synthetic peptides corresponding to one of the variable regions. When applied to sections of the pistil, these antibodies label the intercellular matrix in the stigma and transmitting tissue of the style and the cell walls in the epidermis of the placenta. HindIII digestion of genomic DNA generates a characteristic pattern of S-gene fragments for each genotype. These restriction fragment length polymorphisms can be used to assign S-genotype to progeny arising from breeding experiments.

Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant.

Abstract: The common ice plant is a facultative halophyte in which Crassulacean acid metabolism, a metabolic adaptation to arid environments, can be induced by irrigating plants with high levels of NaCl or by drought. This stress-induced metabolic transition is accompanied by up to a 50-fold increase in the activity of phosphoenolpyruvate carboxylase (PEPCase). To analyze the molecular basis of this plant response to water stress, we have isolated and characterized two members of the PEPCase gene family from the common ice plant. The PEPCase isogenes, designated Ppc1 and Ppc2, have conserved intron-exon organizations, are 76.4% identical at the nucleotide sequence level within exons, and encode predicted polypeptides with 83% amino acid identity. Steady-state levels of mRNAs from the two genes differ dramatically when plants are salt-stressed. Transcripts of Ppc1 increase about 30-fold in leaves within 5 days of salt stress. In contrast, steady-state levels of Ppc2 transcripts decrease slightly in leaf tissue over the same stress period. Steady-state levels of transcripts of both genes decrease in roots over 5 days of salt stress. We have used in vitro transcription assays with nuclei isolated from leaves to demonstrate that the increased expression of Ppc1 caused by water stress occurs in part at the transcriptional level.

Characterization of a pollen-specific cDNA clone from Zea mays and its expression.

Abstract: A pollen-specific cDNA clone, Zmc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 39-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.

Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene.

Abstract: Proteinase inhibitor genes are expressed strongly in specific plant tissues under both developmental and environmental regulation. We have studied the role of the 3' control region of the potato proteinase inhibitor II gene (PI-II) that is inducible in leaves in response to herbivore attacks or other severe wounding. Comparison of the terminator from the PI-II gene with two different terminators from the 6b and 7 genes, driven by a common PI-II promoter-cat fusion molecule, indicated that the PI-II terminator provided the most efficient expression of cat. The PI-II terminator also caused a significantly elevated cat gene expression driven by the cauliflower mosaic virus 35S promoter. The increase in the level of expression is probably not due to the presence of an enhancer element in the PI-II terminator region, but to cis-acting elements involved in mRNA processing or stability. Both transient and stable transformation analyses of the deletion mutants in the 3'-flanking sequence indicated that about a 100-base pair DNA fragment surrounding the polyadenylation site is essential for the efficient gene expression. This region seems to consist of several regulatory elements, including the conserved sequence, CGTGTCTT, which is located 9 bases downstream from the polyadenylation site. The elements appear to contribute to the increased stability of mRNAs containing the PI-II terminator.

Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato.

Abstract: A resistance-breaking strain of tobacco mosaic virus (TMV), Ltb1, is able to multiply in tomatoes with the Tm-2 gene, unlike its parent strain, L. Nucleotide sequence analysis of Ltb1 RNA revealed two amino acid changes in the 30-kD protein: from Cys68 to Phe and from Glu133 to Lys (from L to Ltb1). Strains with these two changes generated in vitro multiplied in tomatoes with the Tm-2 gene and induced essentially the same symptoms as those caused by Ltb1. Strains with either one of the two changes did not overcome the resistance as efficiently as Ltb1, although increased levels of multiplication were observed compared with the L strain. Results showed that both mutations are involved in the resistance-breaking property of Ltb1. Sequence analysis indicated that another resistance-breaking strain and its parent strain had two amino acid changes in the 30-kD protein: from Glu52 to Lys and from Glu133 to Lys. The fact that the amino acid changes occurred in or near the well conserved regions in the 30-kD protein suggests that the mechanism of Tm-2 resistance may be closely related to the fundamental function of the 30-kD protein, presumably in cell-to-cell movement.

Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.

Abstract: We analyzed the expression patterns of several pathogen defense-related genes in primary leaf buds of parsley by in situ RNA hybridization. Labeled antisense RNA probes were generated from seven selected cDNAs detecting transcripts from genes that are rapidly and strongly activated in cultured parsley cells upon treatment with fungal elicitor. These genes encode two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, a furanocoumarin-specific bergaptol O-methyltransferase, one pathogenesis-related protein, and three less well characterized proteins, designated as ELI 3, ELI 5, and ELI 7. In uninfected tissue, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNA levels were high in epidermal cells, oil-duct epithelial cells, and cells of the developing xylem; bergaptol O-methyltransferase mRNA was confined to oil-duct epithelial cells; and the pathogenesis-related protein and ELI 3, ELI 5, and ELI 7 mRNAs were undetectable. All seven mRNAs accumulated transiently and locally around infection sites caused by the soybean-pathogenic fungus Phytophthora megasperma f. sp. glycinea, to which parsley is nonhost-resistant. The observed late appearance of bergaptol O-methyltransferase mRNA, as compared with all other mRNAs, is in accord with a similar relative timing of transient gene activation in elicitor-treated cell cultures. Sharp borders were observed between the infection center, where hypersensitive cell death had occurred in response to fungal penetration, the surrounding area of local gene activation, and the remainder of the tissue not showing any apparent response. Some of the genes were also activated, although less sharply localized, upon wounding of parsley leaves.

Oat Phytochrome Is Biologically Active in Transgenic Tomatoes.

Abstract: To determine the functional homology between phytochromes from evolutionarily divergent species, we used the cauliflower mosaic virus 35S promoter to express a monocot (oat) phytochrome cDNA in a dicot plant (tomato). Immunoblot analysis shows that more than 50% of the transgenic tomato plants synthesize the full-length oat phytochrome polypeptide. Moreover, leaves of light-grown transgenic plants contain appreciably less oat phytochrome than leaves from dark-adapted plants, and etiolated R1 transgenic seedlings have higher levels of spectrally active phytochrome than wild-type tomato seedlings in direct proportion to the level of immunochemically detectable oat polypeptide present. These data suggest that the heterologous oat polypeptide carries a functional chromophore, allowing reversible photoconversion between the two forms of the molecule, and that the far-red absorbing form (Pfr) is recognized and selectively degraded by the Pfr-specific degradative machinery in the dicot cell. The overexpression of oat phytochrome has pleiotropic, phenotypic consequences at all major phases of the life cycle. Adult transgenic tomato plants expressing high levels of the oat protein tend to be dwarfed, with dark green foliage and fruits. R1 transgenic seedlings have short hypocotyls with elevated anthocyanin contents. We conclude that a monocot phytochrome can be synthesized and correctly processed to a biologically active form in a dicot cell, and that the transduction pathway components that interact with the photoreceptor are evolutionarily conserved.

Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome.

Abstract: As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

Binding of a pea nuclear protein to promoters of certain photoregulated genes is modulated by phosphorylation.

Abstract: There have been numerous recent reports documenting phosphorylation of DNA-binding proteins [Montminy and Bilezikjian (1987); Sorger, Lewis, and Pelham (1987); Hoeffler, Kovelman, and Roeder (1988); Jones et al. (1988); Prywes et al. (1988); Sorger and Pelham (1988); Yamamoto et al. (1988)], and the transcriptional regulatory activity of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)]. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of AATATTTTTATT was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and loses all DNA-binding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.

Constitutive expression of pathogenesis-related proteins PR-1, GRP, and PR-S in tobacco has no effect on virus infection.

Abstract: Samsun NN tobacco cells were transformed with chimeric genes for pathogenesis-related (PR) proteins derived from genomic (PR-1a, GRP) or cDNA (PR-S) clones under the transcriptional control of the cauliflower mosaic virus 35S promoter. Regenerated plants were assayed by RNA and protein gel blotting, and plants showing high specific expression of the inserted genes were selected for self-pollination and seed formation. Inspection of second generation transformants showed that constitutive expression of PR-1a, GRP, and PR-S in tobacco in general does not have an effect on the phenotypic appearance of the plants or the expression of other endogenous PR genes. Furthermore, constitutive expression of the above genes does not affect the susceptibility of the plants to infection with tobacco mosaic virus or alfalfa mosaic virus.