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

Author: Sameer Masoud, Published Year: 2002

Planta, 214

Faculty: Science

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.

Keywords: Cell-suspension cultures, cinnamate 4-hydroxylase

Research Title: Altering expression of cinnamic acid 4-hydroxylase in transgenic plants identifies a feedback loop at the entry point into the phenylpropanoid pathway

Author: Sameer Masoud, Published Year: 2000

Plant Physiology , 122

Faculty: Science

Abstract: Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, Lphenylalanine 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.

Keywords: phenylpropanoid pathway, cinnamic acid 4-hydroxylase

Research Title: The corn inhibitor of activated Hageman factor: purification and properties of two recombinant forms of the protein

Author: Sameer Masoud, Published Year: 1998

Expression and Purification , 13

Faculty: Science

Abstract: A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wenet al., Plant Mol. Biol.18, 813–814, 1992) has been expressed inEscherichia coliafter being incorporated into the pT7 expression vector. This inhibitor protein, referred to as CHFI (for the corn inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hageman factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expressed inE. coliin high levels but was insoluble. We solubilized the expressed protein by sonication in 5 M urea and 1% Triton X-100. Several steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per liter of culture). The form with which we have worked most, 7N-CHFI, contains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein indicate it has the expected mass and is properly folded. Functionally, 7N-CHFI is indistinguishable from the inhibitor isolated from corn seeds in its inhibition of porcine trypsin, human β-Factor XIIa, failure to inhibit human plasma kallikrein, and its inhibition of an insect α-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable from 7NCHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect α-amylase. This suggests that the N-terminal region of 7N-CHFI forms at least part of the protein's site of interaction with α-amylase.

Keywords: pT7 expression vector, corn bifunctional inhibitor, Hageman factor

Research Title: Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalnine ammonia-lyase or cinnamate 4-hydroxylase.

Author: Sameer Masoud, Published Year: 1997

Plant Physiology , 115

Faculty: Science

Abstract: We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes Lphenylalanine 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.

Keywords: L-phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, lignin

Research Title: Expression of a corn bifunctional inhibitor of serine proteinase and insect alpha-amylase in transgenic tobacco plants.

Author: Sameer Masoud, Published Year: 1996

Plant Science , 115

Faculty: Science

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.

Keywords: transgenic plants, corn inhibitor

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

Author: Sameer Masoud, Published Year: 1996

Transgenic Research , 5

Faculty: Science

Abstract: cDNA 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.

Keywords: Transgenic alfalfa, Phytophthora megasperma

Research Title: Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco

Author: Sameer Masoud, Published Year: 1994

Bio/technology (Nature Biotechnology), 12

Faculty: Science

Abstract: Plants respond to pathogen attack by the induction of a battery of defenses, suggesting that different protective mechanisms may have complementary roles in the overall expression of disease resistance. We have investigated possible functional interactions between two different hydrolytic enzymes, chitinase and glucanase, by constitutive coexpression in transgenic tobacco of genes encoding the rice RCH10 basic chitinase and the alfalfa AGLU1 acidic glucanase. Hybrid plants were generated by crossing transgenic parental lines exhibiting strong constitutive expression of cauliflower mosaic virus (CaMV) 35S enhancer / RCH10 and CaMV 35S double promoter / AGLU1 gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous for each transgene, and in homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone. Productive interactions between chitinase and glucanase transgenes in vivo point to combinatorial expression of antimicrobial genes as an effective approach to engineering enhanced crop protection against fungal disease.

Keywords: hydrolytic enzymes, antimicrobial, engineering crop protection

Research Title: Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants.

Author: Sameer Masoud, Published Year: 1993

Plant Molecular Biology, 21

Faculty: Science

Abstract: 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 microg/g) than in roots (30microg/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to antirOC antibodies. OC-I from both sources was active against papain.

Keywords: Agrobacterium tumefaciens, 35S promoter, transgenic tobacco

Research Title: The sequence within two primers influences the optimum concentration of dimethyl sulfoxide in the PCR

Author: Sameer Masoud, Published Year: 1992

PCR Methods and Applications , 2

Faculty: Science

Abstract: We used the polymerase chain reaction (PCR) to examine extracts of transgenic tobacco plants for the presence of foreign DNA sequences. During this work, information regarding the effect of dimethyl sulfoxide (DMSO) in the PCR was obtained.

Keywords: DMSO, PCR

Research Title: C-banding of alfalfa chromosomes: standard karyotype and analysis of a somaclonal variant.

Author: Sameer Masoud, Published Year: 1991

Journal of Heredity , 82

Faculty: Science

Abstract: C-banding was used to identify diploid alfalfa (Medicago sativa L. cv. CADL; 2n = 2x = 16) somatic chromosomes and to construct a karyogram. CADL is “cultivated alfalfa at the diploid level” and was derived from cultivated tetraplolds. All chromosomes were differentially C-banded and were differentially C-banded and were identified individually based on position, number, and/or staining intensity of bands. All chromosomes showed centromeric band(s). Six of eight chromosomes—1, 2, 4, 5, 6, and 8—had telomeric bands but differed in number (0–2) and position of intercalary bands. Chromosomes 3 and 7, which lacked telomeric bands, were also distinguishable. Chromosome 3 exhibited an intercalary band, whereas chromosome 7 was smaller and possessed only a centromeric band. C-banding polymorphism was observed for CADL within some homologous chromosome pairs. The usefulness and reproducibility of the C-banding technique was demonstrated by the analysis of a plant regenerated from tissue culture. Chromosomal rearrangements were documented, including an apparent deletion and an isochromosome derived from chromosome 8. It should now be feasible to use C-banding to gain basic cytogenetic information and to monitor chromosome transfers from other Medicago species into alfalfa.

Keywords: C-banding, alfalfa, Chromosomal rearrangements ,