Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

Developmental and light-regulated post-translational control of 3-hydroxy-3-methylglutaryl-CoA reductase levels in potato.

In plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). We have added an eight amino-acid-residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR-FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR-FLAG protein were found only in apical meristematic tissue, suggesting post-translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR-FLAG transgenic and the non-transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post-translational level. After 2.5 h in darkness, levels of HMGR-FLAG are approximately one-half of those in plants in the light; protein levels recover rapidly when dark-treated plants are returned to the light. In non-transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR-FLAG remain associated with polyribosomes in dark-treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR-FLAG. The epitope-tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light-regulated control of HMGR occurs post-translationally in vivo.

Trade-offs between pathogen and herbivore resistance.

During the past year genetic and pharmacological experiments have revealed a molecular basis for the cross-talk between signaling pathways mediating pathogen and herbivore resistance. These findings provide considerable insight into the apparently contradictory results reported for trade-offs between pathogen and herbivore resistance.

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway.

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

HMG-CoA reductase gene families that differentially accumulate transcripts in potato tubers are developmentally expressed in floral tissues.

We isolated two full-length cDNA clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from potato (Solanum tuberosum) L. tubers. The clones, designated hmg2.2 and hmg3.3, are members of previously described gene subfamilies. In addition to being induced by arachidonic acid in tubers, hmg2.2 transcript accumulates developmentally in young flowers, and in mature sepals and ovaries, whereas transcript for hmg3.3 accumulates in mature petals and anthers. Our data suggest that members of specific HMGR-encoding gene subfamilies might be involved in both defense responses and flower development. Accumulation of different HMGR transcripts could provide some control of isoprenoid biosynthesis by producing isoforms specific for classes of end-products produced in particular tissues.

Evidence for Chewing Insect-Specific Molecular Events Distinct from a General Wound Response in Leaves.

The timing of transcript accumulation of several wound-induced genes is different in insect-damaged and mechanically damaged leaves. Transcripts for the proteinase inhibitor II and 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes accumulate more rapidly in potato (Solanum tuberosum L.) leaves chewed on by caterpillars than in leaves damaged mechanically. The timing of maximum transcript accumulation was not affected by the degree of damage inflicted by the insect larvae. When applied to a mechanical wound site, regurgitant isolated from Manduca sexta larvae causes transcript accumulation profiles to shift to parallel those in insect-damaged tissue. Whether obtained from larvae fed either potato leaves or a nonplant diet, insect regurgitant fed through the petiole of detached leaves also induces accumulation of these transcripts. The transcript accumulation-inducing activity of regurgitant is enhanced by heating at 100[deg]C. Our data suggest that a heat-stable, insect-derived elicitor functions to induce the rapid accumulation of transcripts that may be involved in plant defense against herbivores. Distinct signal transduction pathways that can distinguish between insect damage and abiotic damage might therefore exist in plants.

Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures).

Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Regulation of HMG-CoA reductase activity in plants.

This brief review summarizes the current literature on the regulation of HMG-CoA reductase (HMGR) in plants. The mevalonate pathway, which starts with the synthesis of mevalonate by HMGR, has more branch pathways in plants than in most other organisms, leading to a tremendous variety of isoprenoid products. Evidence suggests that HMGR is an important control point for the synthesis of many of these plant isoprenoids, including some that are vital for primary metabolism and pest resistance. Plant HMGR activity responds in vivo to a variety of developmental and environmental signals, such as cell division, light, and infection. Plants regulate HMGR activity at the level of mRNA by differential induction of HMGR gene family members, and posttranslationally by enzyme modification. Calcium, calmodulin, and proteolytic degradation may also have a role in regulation of plant HMGR.

Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

URF13, a maize mitochondrial pore-forming protein, is oligomeric and has a mixed orientation in Escherichia coli plasma membranes.

URF13, an inner mitochondrial membrane protein of the maize Texas male-sterile cytoplasm (cms-T), has one orientation in the inner membrane of maize mitochondria but two topological orientations in the plasma membrane when expressed in Escherichia coli. Antibodies specific for the carboxyl terminus of URF13 and for an amino-terminal tag fused to URF13 in E. coli were used to determine the location of each end of the protein following protease treatments of right-side-out and inside-out vesicles derived from cms-T mitochondria and the E. coli plasma membrane. Cross-linking studies indicate that a portion of the URF13 population in mitochondria and E. coli exists in membranes in an oligomeric state and, in combination with proteolysis studies, show that individual subunits within a given multimer have the same orientation. A three-membrane-spanning helical model for URF13 topology is presented.