Sameer Masoud

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review.

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants

Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases. A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3′ region was introduced into tobacco plants by Agrobacterium tumefaciens. The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis. Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli. Similar bands were absent in extracts from untransformed control plants. OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny. On a fresh weight basis, the OC-I content was higher in leaves (50 μg/g) than in roots (30 μg/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies. OC-I from both sources was active against papain.

Constitutive expression of an inducible β-1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f.sp. medicaginis, but does not reduce disease severity of chitin-containing fungi.

AbstractcDNA sequences coding for an acidic glucanase (Aglul) that is expressed in elicited alfalfa cell suspension cultures, and a rice basic chitinase (RCH10) that is induced by elicitor and wounding, were placed into constitutive expression cassettes under control of the cauliflower mosaic virus 35S promoter or 35S enhancer sequences, and introduced in alfalfa plants of the regenerable cultivar Regen SY byAgrobacterium-mediated transformation. Southern and northern blot analysis confirmed stable incorporation and transcription, respectively, of the chimaeric genes in the transgenic plants. Active rice chitinase was expressed in alfalfa leaves, and leaves of plants transformed with theAglul sequence exhibited increased glucanase activity and the appearance of an additional glucanase band on activity gels. A glucanase of similar native electrophoretic mobility was constitutively present in root extracts of non-transformed alfalfa plants, and was induced in pathogen-infected leaves, presumably reflecting the expression pattern of the endogenousAglul gene. Thus, expression of the chimaericAglul transgene increased the amount, and broadened the tissue-type constitutive expression, of theAglul protein compared to control plants. Transgenic alfalfa plants containing a binary vector with both chimaeric genes in tandem expressed each gene to a much lesser extent than transgenic plants containing a single chimaeric gene. Expression of RCH10 in transgenic alfalfa did not appear to affect negatively theRhizobium/alfalfa interaction. Analysis of primary transformants for response to several fungal pathogens of alfalfa indicated statistically significant symptom reduction only in the case ofPhytophthora megasperma f. sp.medicaginis (Pmm), and only in plants overexpressingAglul. Resistance against Pmm segregated with glucanase expression in a cross between transgenic Regen SY and the commercial alfalfa cultivar Apollo.

Over-expression of cinnamate 4-hydroxylase leads to increased accumulation of acetosyringone in elicited tobacco cell-suspension cultures

Abstract. Cell-suspension cultures were produced from transgenic tobacco (Nicotiana tabacum L. ) plants harboring a constitutively expressed alfalfa cinnamate 4-hydroxylase (C4H) transgene. Increased levels of C4H enzyme activity in the transgenic cultures were observed only following exposure of the cells to yeast elicitor, although alfalfa C4H transcripts were expressed at a high level from the cauliflower mosaic virus 35S promoter in the absence of elicitation. Increased expression of C4H in elicited cell-suspension cultures had no appreciable effect on the HPLC profiles of soluble phenolic compounds. However, levels of one compound, subsequently identified as 3,5-dimethoxy-4-hydroxy acetophenone (acetosyringone), were strongly elevated in the wall-bound phenolic fraction. The results are discussed in relation to the correlation between C4H activity and the synthesis of 3,5-dimethylated hydroxycinnamic acid derivatives in tobacco.

Virulence of root-knot nematodes, Meloidogyne spp., on tomato bearing the Mi gene for resistance

Three species of root-knot nematodes, Meloidogyne javanica, M. incognita races 1 and 2, and M. arenaria race 2 occur in Jordan. These species and their races were identified using morphological characters, the North Carolina differential host test, and SCAR-PCR. The virulence of 83 isolates belonging to Meloidogyne species and races was assayed. The virulence assay was based on the isolate reproduction rate on a resistant tomato cultivar Betterboy bearing the Mi gene for resistance, and was compared with that on the susceptible tomato cultivar Rutgers. Three M. javanica isolates were highly virulent on the resistant cv. Betterboy as indicated by their high root gall index (4.73) and high reproduction factor (3.73). The horticultural parameters (shoot and root fresh weights and root dry weight) were negatively correlated with the reproduction factor.

Expression of a corn bifunctional inhibitor of serine proteinases and insect α-amylases in transgenic tobacco plants

Abstract Corn seed endosperm contains a putative defense protein (14K-CI) that inhibits both trypsin-like serine proteinases and insect α-amylases. A cDNA clone encoding 14K-CI under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase polyadenylation region was introduced into tobacco plants by Agrobacterium -mediated transformation. The presence and expression of the chimeric gene in regenerated (R0) and progeny (R1) plants was confirmed by Southern, polymerase chain reaction, and Northern analyses. Protein extracts of selected transformed plants, but not control plants, reacted positively to corn seed 14K-CI antiserum. Comparison of 14K-CI mobilities from transgenic leaves and corn seeds after denaturing polyacrylamide gel electrophoresis indicated that recognition and cleavage of the 14K-CI signal peptide sequence occurred in tobacco leaves. Immunological assays showed that the inhibitor was expressed in amounts up to 0.05% of the total protein in young leaves of R1 plants. Lesser accumulation was generally detected in older leaves. Protein extracts from transgenic plants were more inhibitory than were control plant extracts to bovine trypsin activity. Four tobacco plants with a gene lacking the 14K-CI signal peptide region accumulated 3–5-fold less inhibitor than did the highest-expressing plant with the full cDNA clone. Use of a double 35S promoter did not enhance 14K-CI accumulation. Post-transcriptional events appear to be a major factor in the low accumulation of 14K-CI in tobacco.

Overexpression of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in tobacco cell suspension cultures

Cell suspension cultures were generated from transgenic tobacco plants expressing bean phenylalanine ammonia-lyase (PAL) or alfalfa cinnamate 4-hydroxylase (C4H) transgenes. PAL over-expressing cell lines exhibited high basal levels of PAL transcripts and enzymatic activity, but hardly exceeded wild-type cultures in the level of PAL activity obtained following elicitation. In contrast, expression of alfalfa C4H transcripts in unelicited cell suspension cultures did not result in a corresponding increase in C4H enzymatic activity. However, following elicitation, the level of C4H activity in over-expressing lines was significantly greater than in control lines.