Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana.

Cell-specific expression patterns of the Eucalyptus gunnii cinnamoyl coenzymeA reductase (EgCCR) and cinnamyl alcohol dehydrogenase (EgCAD2) promoters were analyzed by promoter-GUS histochemistry in the primary and secondary xylem tissues from floral stems and roots of Arabidopsis thaliana. Expression patterns indicated that the EgCCR and EgCAD2 genes were expressed in a coordinated manner in primary and secondary xylem tissues of the Arabidopsis floral stem and root. Both genes were expressed in all lignifying cells (vessel elements, xylem fibers and paratracheal parenchyma cells) of xylem tissues. The capacity for long-term monolignol production appeared to be related to the cell-specific developmental processes and biological roles of different cell types. Our results suggested that lignification of short-lived vessel elements was achieved by a two-step process involving (i) monolignol production by vessel elements prior to vessel programmed cell death and (ii) subsequent monolignol production by vessel-associated living paratracheal parenchyma cells following vessel element cell death. EgCCR and EgCAD2 gene expression patterns suggested that the process of xylem cell lignification was similar in both primary and secondary xylem tissues in Arabidopsis floral stems and roots.

Plant glycoside hydrolases involved in cell wall polysaccharide degradation.

The cell wall plays a key role in controlling the size and shape of the plant cell during plant development and in the interactions of the plant with its environment. The cell wall structure is complex and contains various components such as polysaccharides, lignin and proteins whose composition and concentration change during plant development and growth. Many studies have revealed changes in cell walls which occur during cell division, expansion, and differentiation and in response to environmental stresses; i.e. pathogens or mechanical stress. Although many proteins and enzymes are necessary for the control of cell wall organization, little information is available concerning them. An important advance was made recently concerning cell wall organization as plant enzymes that belong to the superfamily of glycoside hydrolases and transglycosidases were identified and characterized; these enzymes are involved in the degradation of cell wall polysaccharides. Glycoside hydrolases have been characterized using molecular, genetic and biochemical approaches. Many genes encoding these enzymes have been identified and functional analysis of some of them has been performed. This review summarizes our current knowledge about plant glycoside hydrolases that participate in the degradation and reorganisation of cell wall polysaccharides in plants focussing particularly on those from Arabidopsis thaliana.

Genetic variations of cell wall digestibility related traits in floral stems of Arabidopsis thaliana accessions as a basis for the improvement of the feeding value in maize and forage plants.

Floral stems of Arabidopsis thaliana accessions were used as a model system relative to forage plant stems in genetic variation studies of lignin content and cell wall digestibility related traits. Successive investigations were developed in a core collection of 24 Arabidopsis accessions and in a larger collection of 280 accessions. Significant genetic variation for lignin content in the cell wall, and for the two in vitro cell wall digestibility investigated traits, were found both in the core collection and in the large collection. Genotype x environment interactions, investigated in the core collection, were significant with a few genotypes contributing greatly to interactions, based on ecovalence value estimates. In the core collection, genotypes 42AV, 224AV, and 8AV had low cell wall digestibility values, whatever be the environmental conditions. Genotype 157AV, observed only in one environment, also appeared to have a low cell wall digestibility. Conversely, genotypes 236AV, 162AV, 70AV, 101AV, 83AV had high cell wall digestibility values, genotype 83AV having a slightly greater instability across differing environments than others. The well-known accession Col-0 (186AV) appeared with a medium level of cell wall digestibility and a weak to medium level of interaction between environments. The ranges of variation in cell wall digestibility traits were higher in the large collection than in the core collection of 24 accessions, these results needing confirmation due to the lower number of replicates. Accessions 295AV, 148AV, and 309AV could be models for low stem cell wall digestibility values, with variable lignin content. Similarly, accessions 83AV and 162AV, already identified from the study of the core collection, and five accessions (6AV, 20AV, 91AV, 114AV, and 223AV) could be models for high stem cell wall digestibility values. The large variations observed between Arabidopsis accessions for both lignin content and cell wall digestibility in floral stems have strengthened the use this species as a powerful tool for discovering genes involved in cell wall biosynthesis and lignification of dicotyledons forage plants. Investigations of this kind might also be applicable to monocotyledons forage plants due to the basic similarity of the genes involved in the lignin pathway of Angiosperms and the partial homology of the cell wall composition and organization of the mature vascular system in grasses and Arabidopsis.

Potential effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae).

Protease inhibitors (PIs) have been shown to cause lethal and sublethal effects on aphids depending on the kind of PI and aphid species. Therefore, these proteins might affect aphid parasitoids directly by inhibiting their digestive proteolysis or indirectly via their development in a less suitable host. In our study, the risk of exposure and the potential effects of soybean Bowman-Birk inhibitor (SbBBI) and oryzacystatin I (OCI) on the aphid endoparasitoid Aphidius ervi were investigated using artificial diet to deliver PIs. Immunoassays showed that both SbBBI and OCI were detected in the honeydew of aphids reared on artificial diet containing these recombinant proteins at 100 microg/mL. However, only SbBBI was detected in parasitoid larvae, while this PI could not be detected in adult parasitoids emerged from PI-intoxicated aphids. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of A. ervi predominantly relies on serine proteases and especially on chymotrypsin-like activity. Bioassays using SbBBI and OCI on artificial diet were performed. A. ervi that developed on intoxicated aphids had impaired fitness. Thus development and parasitism success of parasitoids exposed to OCI were severely affected. On the contrary, SbBBI only altered significantly female size and sex ratio. Direct exposure to PIs through adult food intake did not affect female's longevity, while SbBBI and OCI (100 microg/mL) induced 69% and 30% inhibition of digestive protease activity, respectively. These studies made it possible to estimate the risk of exposure to plant PIs and the sensitivity of the aphid parasitoid A. ervi to these entomotoxins, by combining immunological, biochemical and biological approaches. First it pointed out that only immature stages are affected by PIs. Secondly, it documented two different modes of effect, according to the nature of the PIs and both host and parasitoid susceptibility. OCI prevented the development of A. ervi mainly due to the host susceptibility, whereas SbBBI only induced sublethal effects on the parasitoid, possibly due to both direct action on the parasitoid susceptible proteases, and host-mediated action through size reduction.

Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae).

Transgenic plants expressing protease inhibitors (PIs) have emerged in recent years as an alternative strategy for pest control. Beneficial insects such as parasitoids may therefore be exposed to these entomotoxins either via the host or by direct exposure to the plant itself. With the objective of assessing the effects of PIs towards aphid parasitoids, bioassays using soybean Bowman-Birk inhibitor (SbBBI) or oryzacystatin I (OCI) on artificial diet were performed on Macrosiphum euphorbiae-Aphelinus abdominalis system. OCI significantly reduced nymphal survival of the potato aphid M. euphorbiae and prevented aphids from reproducing. This negative effect was much more pronounced than with other aphid species. On the contrary, SbBBI did not affect nymphal viability but significantly altered adult demographic parameters. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of Aphelinus abdominalis predominantly relies on serine proteases and especially on chymotrypsin-like activity. Immunoassays suggested that OCI bound to aphid proteins and accumulated in aphid tissues, whereas SbBBI remained unbound in the gut. Bioassays using M. euphorbiae reared on artificial diets supplemented with both OCI and SbBBI showed a fitness impairment of Aphelinus abdominalis that developed on intoxicated aphids. However, only SbBBI was detected in parasitoid larvae, while no PI could be detected in adult parasitoids that emerged from PI-intoxicated aphids. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed.

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii.

We have characterized proteinase activities in gut extracts from the cotton-melon aphid (Aphis gossypii Glover), an insect feeding strictly on protein-poor phloem. The major, if not exclusive, intestinal proteinases of this aphid are of the cysteine type. A cDNA has been cloned from a gut library and codes for the cysteine proteinase AgCatL, a cathepsin L-like cysteine proteinase. The AgCatL protein shows high sequence similarity with mammalian and some arthropod cathepsin L-like proteinases, but can be reliably distinguished from the secreted (digestive) proteinases identified in other arthropods. AgCatL is widely expressed in aphid intestinal cells. Immunolocalization of AgCatL showed an intense signal at the level of the anterior 'stomach' part of the midgut, and especially at intracellular localization. Although the precise role of AgCatL in aphid midgut physiology is still unclear, this enzyme could be involved in the processing of exogenous ingested polypeptides.

Impact of oilseed rape expressing the insecticidal cysteine protease inhibitor oryzacystatin on the beneficial predator Harmonia axyridis (multicoloured Asian ladybeetle).

Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) on the predatory ladybird Harmonia axyridis were investigated using diamondback moth Plutella xylostella as the pest species. As expected, oilseed rape expressing OC-1 had no effects on either development or survival of the pest, which utilizes serine digestive proteases. Immunoassays confirmed accumulation of the transgene product in pest larval tissues at levels of up to 3 ng per gut. Characterization of proteolytic digestive enzymes of H. axyridis demonstrated that larvae and adults utilize cysteine and aspartic proteases; the former activity was completely inhibited by oryzacystatin in vitro. However, when H. axyridis larvae consumed prey reared on OC-1 expressing plants over their entire life cycle, no significant effects upon survival or overall development were observed. The inhibitor initially stimulated development, with a shortening of the developmental period of the second instar by 27% (P < 0.0001) accompanied by a 36% increase in weight of second instar larvae (P = 0.007). OC-1 had no detrimental effects on reproductive fitness of adult H. axyridis. Interestingly there was a significant increase in consumption of OC-1 dosed prey. The results show that prey reared on transgenic plants expressing a protein which inhibited ladybird digestive enzymes in vitro had no effects in vivo; the ladybird was able to up-regulate digestive proteases in response to the inhibitor.

Population-scale laboratory studies of the effect of transgenic plants on nontarget insects.

Studies of the effects of insect-resistant transgenic plants on beneficial insects have, to date, concentrated mainly on either small-scale "worst case scenario" laboratory experiments or on field trials. We present a laboratory method using large population cages that represent an intermediate experimental scale, allowing the study of ecological and behavioural interactions between transgenic plants, pests and their natural enemies under more controlled conditions than is possible in the field. Previous studies have also concentrated on natural enemies of lepidopteran and coleopteran target pests. However, natural enemies of other pests, which are not controlled by the transgenic plants, are also potentially exposed to the transgene product when feeding on hosts. The reduction in the use of insecticides on transgenic crops could lead to increasing problems with such nontarget pests, normally controlled by sprays, especially if there are any negative effects of the transgenic plant on their natural enemies. This study tested two lines of insect-resistant transgenic oilseed rape (Brassica napus) for side-effects on the hymenopteran parasitoid Diaeretiella rapae and its aphid host, Myzus persicae. One transgenic line expressed the delta-endotoxin Cry1Ac from Bacillus thuringiensis (Bt) and a second expressed the proteinase inhibitor oryzacystatin I (OC-I) from rice. These transgenic plant lines were developed to provide resistance to lepidopteran and coleopteran pests, respectively. No detrimental effects of the transgenic oilseed rape lines on the ability of the parasitoid to control aphid populations were observed. Adult parasitoid emergence and sex ratio were also not consistently altered on the transgenic oilseed rape lines compared with the wild-type lines.

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction.

Transcription analysis of a mustard (Sinapis alba L.) serine proteinase inhibitor gene revealed identical 5' termini of mRNAs synthesized during seed maturation and chemical or wounding induction. Polyadenylation of mRNAs on multiple or single sites differentiated gene expression, increasing the availability of stable mRNAs during seed maturation compared with chemical and wounding induction. Expression of the beta-glucuronidase (GUS)-encoding region of the UidA reporter gene, detected under the control of deleted segments of the region flanking on the 5' side the mit-2 gene, identified a stretch of about 520 bp essential for gene expression. The presence in this region of two ABRE motifs is relevant for plant response to gene induction. Expression of GUS was detectable under different induction stimuli in several organs such as seedlings and leaves and was active to varying extents in the vascular tissues and meristem.

NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars.

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.

Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests.

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.

Regulation of sulfur nutrition in wild-type and transgenic poplar over-expressing gamma-glutamylcysteine synthetase in the cytosol as affected by atmospheric H2S.

This study with poplar (Populus tremula x Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing gamma-glutamylcysteine (gamma-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H2S. H(2)S taken up by the leaves increased cysteine, gamma-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H2S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and gamma-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H(2)S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.

Isolation and characterization of a dehydrin gene from white spruce induced upon wounding, drought and cold stresses.

A cDNA clone encoding a dehydrin gene was isolated from a cDNA library prepared from white spruce (Picea glauca) needle mRNAs. The cDNA, designated PgDhn1, is 1159 nucleotides long and has an open reading frame of 735 bp with a deduced amino acid sequence of 245 residues. The PgDhn1 amino acid sequence is highly hydrophilic and possesses four conserved repeats of the characterized lysine-rich K-segment (EKKGIMD-KIKEKLPG), and an 8-serine residue stretch prior to the first lysine-rich repeat that is common to many dehydrins. The DEYGNP conserved motif is, however, absent in the PgDhn1 sequence. In unstressed plants, the highest level of transcripts was detected in stem tissue and not fully expanded vegetative buds. PgDhn1 expression was also clearly detected in reproductive buds, at various stages of development. The mRNAs corresponding to PgDhn1 cDNA were induced upon wounding and by jasmonic acid (JA) and methyl jasmonate (MeJa) treatments. Upon drought stress, increased transcript accumulation was observed in needle tissue reaching a maximum level 48 h after treatment. Treatments of seedlings with abscisic acid or ethephon also resulted in high levels of transcript accumulation in needle tissue. Finally, cold induction of PgDhn1 transcripts was also detected as early as 8 h after treatment.

Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity.

Transgenic poplars (Populus tremula x Populus alba) were obtained by introduction of a sense homologous transgene encoding caffeic acid O-methyltransferase (COMT) under the control either of the cauliflower mosaic virus double 35S promoter or of the eucalyptus cinnamyl alcohol dehydrogenase promoter. Although these constructs conferred a moderate overexpression of COMT in some lines, a transgenic line with the double 35S promoter was found where COMT activity in woody tissues was close to zero due to a gene-silencing phenomenon. For the first time in COMT down-regulated trees, this alteration substantially reduced lignin level in 6-month-old trees (17% decrease). Lignin structure was found to be strongly altered, with a two times higher content in condensed bonds, an almost complete lack of syringyl units, and the incorporation of 5-hydroxyguaiacyl units to the most remarkable extent reported so far. Consistent with the higher cellulose content and with the higher condensation degree of the lignin, the impact of the transformation on the kraft-pulping performances of the poplar trees positively affected the pulp yield (10% relative increase), but made lignins less amenable to industrial degradations.

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis.

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.

Isolation and characterization of a cDNA clone encoding a putative white spruce glycine-rich RNA binding protein.

A presumably full-length cDNA encoding a putative glycine-rich RNA binding protein was isolated from a lambdaZAP cDNA library prepared from mRNAs extracted from needles of 2year old white spruce seedlings, which had been either wounded or jasmonate-treated. The cDNA, designated PgRNP (Picea glauca RNP protein), presents a 468bp open reading frame encoding a 155 amino acid protein. This polypeptide possesses an RNA binding domain (RNP-CS) and a glycine-rich domain. Comparative alignment reveals extensive homologies to glycine-rich RNA binding proteins containing an RNP-CS found in other angiosperm species. Genomic hybridization experiments suggest that the PgRNP gene is part of a small multigene family with at least four members. RNA blot analysis revealed that the PgRNP transcript is expressed in all tissues from non-stressed plants. Constitutive mRNA level was found in needle tissue from control as well as methyl-jasmonate treated plants. Wounding had no clear induction effect. Jasmonic acid treatment and systemic wound response had a positive effect on transcript accumulation. Transcript accumulation was slightly induced by cold in needles, and repressed by drought stress in both needle and root tissues of 2year old plants. Finally, the level of PgRNP accumulation was induced by wounding and repressed in 2week old dark-grown seedlings upon jasmonate treatments.

Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase.

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.

Molecular cloning of cDNAs encoding a range of digestive enzymes from a phytophagous beetle, Phaedon cochleariae.

To gain better knowledge of the variety of digestive enzymes in phytophagous coleopteran pests, a sequencing screen of 76 random cDNAs from a gut library from Phaedon cochleariae larvae was performed. The screen yielded 21 cDNAs encoding amino-acid sequences homologous to known digestive enzymes, most of them were cell wall-hydrolysing enzymes. The deduced protein sequences of 7 cDNAs encoding putative alpha-amylase, cysteine proteinase, trypsin, chymotrypsin, cellulase, pectinase and xylanase display all the structural features that characterize these enzymes in other eukaryotic organisms. Except the alpha-amylase and chymotrypsin cDNAs, the other cDNAs probably derive from multigene families. The distribution of the corresponding enzymatic activities at various developmental stages of P. cochleariae was examined. alpha-amylase activity is present in guts of larvae and adults, proteinases are abundant in guts of larvae and adults, but scarce in eggs and larval carcasses, xylanases are present in the guts of larvae and adults, as well as in carcasses of larvae, whereas cellulase and pectinase activities are distributed in larval and adult guts, larval carcasses, and eggs. Only a minor fraction of the cellulases is secreted by microorganisms, suggesting that P. cochleariae synthesizes most of its own cell-wall hydrolysing enzymes. The physiological role of the enzymes is discussed, as well as the significance of these results for pest management strategies involving transgenic plants expressing enzyme inhibitors.