M. S. Srinivasa Reddy

The phenylpropanoid pathway and plant defence—a genomics perspective

Summary The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex flavonoids, isoflavonoids, and stilbenes. The enzymatic steps involved in the biosynthesis of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned. Less is understood about the regulatory genes that orchestrate rapid, coordinated induction of phenylpropanoid defences in response to microbial attack. Many of the biosynthetic pathway enzymes are encoded by gene families, but the specific functions of individual family members remain to be determined. The availability of the complete genome sequence of Arabidopsis thaliana, and the extensive expressed sequence tag (EST) resources in other species, such as rice, soybean, barrel medic, and tomato, allow, for the first time, a full appreciation of the comparative genetic complexity of the phenylpropanoid pathway across species. In addition, gene expression array analysis and metabolic profiling approaches make possible comparative parallel analyses of global changes at the genome and metabolome levels, facilitating an understanding of the relationships between changes in specific transcripts and subsequent alterations in metabolism in response to infection.

Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to ∼65% P (from wild-type levels of ∼1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key β-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of β-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range13C-1H correlation experiments. For example, although β-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.

Recent Advances in Soybean Transformation

Soybean (Glycine max (L.) Merrill) transformation continues to be problematic even with the efforts of numerous researchers in the field. In spite of this low efficiency, soybean transformation is now routine utilizing the somatic embryogenie and meristematic tissue culture methods. A comparison, including the advantages and disadvantages, of each method are presented in this review. Soybean transformation is currently done using both Agrobacterium tumefaciens and particle bombardment. We review some of the current selectable marker genes in use, and we also describe additional non-standard transformation techniques that have been, or are being attempted, to produce transgenic soybeans.

Gene silencing in transgenic soybean plants transformed via particle bombardment

Transgenes are susceptible to silencing in plants especially when multiple copies of the gene of interest are introduced. Transgenic plants derived by particle bombardment, which is the common method for transforming soybean, have a tendency to have multiple integration events. Three independent transgenic soybean plants obtained via particle bombardment were analyzed for transgene silencing. A GUS transgenic soybean line had at least 100 copies of the GUS gene while there were approximately 60 copies of the transgene in the two soybean lines transformed with a 15-kDa zein storage protein gene from maize. Soybean plants transformed with the GUS gene showed variable GUS expression. The coding region and promoter of the GUS gene in the plants with low expression of GUS were heavily methylated. Variability in GUS expression was observed in the progeny of the high expressors in the T2 and T3 generations as well. Expression level of the 15-kDa zein gene in transgenic soybean plants showed correlation with the level of transgene methylation. The helper component-proteinase from potyviruses is known to suppress post-transcriptional gene silencing. Transgenic plants were inoculated with the soybean mosaic potyvirus (SMV) to test possible effects on transgene silencing in soybean. Infection with SMV did not suppress transgene silencing in these plants and suggests that the silencing in these plants may not be due to post-transcriptional gene silencing.

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus.

Overexpression of the Arabidopsis thaliana MinE1 bacterial division inhibitor homologue gene alters chloroplast size and morphology in transgenic Arabidopsis and tobacco plants.

Abstract. Higher-plant chloroplast division requires some of the same genes that are involved in prokaryotic cell division. These include the FtsZ and MinD proteins. Other genes that might be involved in higher-plant chloroplast division have yet to be characterized. The Arabidopsis thaliana (L.) Heynh. MinE (AtMinE1) gene was identified in the genomic database, isolated by reverse transcription-polymerase chain reaction and constitutively expressed in tobacco (Nicotiana tabacum L.) and Arabidopsis plants in both the sense and antisense orientation. Confocal and electron-microscopic analysis of the sense-overexpressing AtMinE1 transgenic tobacco and Arabidopsis plants revealed that the chloroplasts were abnormal in size and shape compared to wild-type Arabidopsis and tobacco chloroplasts. Our results, based on the overexpression of the AtMinE1 gene in tobacco and Arabidopsis, confirm that the AtMinE1 gene is involved in plant chloroplast division.

INCREASED SULFUR AMINO ACIDS IN SOYBEAN PLANTS OVEREXPRESSING THE MAIZE 15 kDa ZEIN PROTEIN

SummaryFour transgenic soybean [Glycine max (L.) Merrill] lines were generated containing the maize 15 kDa zein protein gene using somatic embryogenic protocols. The zein gene was inserted behind the β-phaseolin promoter for seed-specific expression. All four lines represent separate transformation events as they were generated in different experiments at different locations. Two of the transformation events produced multiple plants, and these produced identical Southern hybridization patterns (UKY/Z1, UKY/Z2 and UKY/Z3 from the first; and OSU/Z4, OSU/Z8 and OSU/Z10 from the second). Molecular characterization revealed that multiple copies of the zein gene were present in all of the transgenic lines. Immunoblot analysis confirmed the accumulation of the zein protein product in the seeds of the UKY/Z1, UKY/Z2, UKY/Z3, OSU/Z4, OSU/Z8 and OSU/Z10 transgenic lines. However, there was no accumulation of zein protein in the UGA/Z1 line and Northern analysis confirmed that the zein transgene was silenced in this line. It was not possible to analyze the zein expression in the seeds of the UKY/Z4 line, as it was sterile. Amino acid analysis of the UKY and OSU lines confirmed that there was a 12–20% increase in methionine, and 15–35% increase in cysteine content in these lines compared to the control. There were no consistent changes in the content of the other amino acids in the transgenic lines. These results suggest that while the increase in methionine content in these lines is modest, it is possible to increase the methionine content without adversely affecting the protein composition of soybean.

Resistance to Bean pod mottle virus in Transgenic Soybean Lines Expressing the Capsid Polyprotein

ABSTRACT Transgenic fertile soybean plants were generated from somatic embryos of soybean (Glycine max) cv. Jack transformed via particle bombardment with the capsid polyprotein (pCP) gene of Bean pod mottle virus(BPMV). The plant transformation vector (pHIG/BPMV-pCP) utilized in these experiments contained the BPMV-pCP coding sequence, an intron-containing GUS gene, and the hygromycin phosphotransferase gene. Southern blot hybridization analysis showed that 19 transgenic soybean plants selected for resistance to hygromycin contained the genes for GUS and BPMV-pCP. The progeny of five of these transgenic soybean plants (plants 137, 139, 157, 183, and 186) were characterized in detail. An additional transgenic plant (plant 200) contained the intron-GUS and hygromycin resistance genes, but lacked the BPMV-pCP gene and was used as a negative control. Southern blot hybridization analysis of the five transgenic plants showed the presence of three copies of the T-DNA in a similar banding pattern suggesting that they were derived from a single transformation event. Western and northern blot analyses showed that the expression levels of BPMV-pCP and pCP transcript were high in these five pCP plants. Infectivity assays with detached leaves demonstrated that all five pCP plants exhibited resistance to virus infection because they accumulated lower levels of BPMV compared with plant 200 and nontransformed controls. Unlike the T(2) progeny of line 183-1 that segregated with respect to the pCP gene and, consequently, to BPMV resistance, the T(2) progeny of the homozygous line 183-2 showed little or no symptoms in response to rub-inoculation with virions of a severe strain of BPMV. Although BPMV accumulation was evident in leaves on which viruliferous beetles were allowed a 72-h inoculation access period, the upper noninoculated leaves of the T(2) progeny of line 183-2 plants were symptomless and accumulated little or no virus. Because the progeny of this homozygous transgenic line exhibited systemic resistance, they could potentially be useful in generating commercial cultivars resistant to BPMV.

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants.

Abstract. The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the ArabidopsisMinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.

Analysis of the Agrobacterium tumefaciens pTiChry5 6b Promoter

The 6b gene of Agrobacterium tumefaciens has evolved to become transcriptionally active in plant cells and has been postulated to modify the activity of plant growth regulators, auxins and cytokinins. To delimit the region that is necessary for expression and is responsible for auxin inducibility, we have constructed a series of 5′ deletions and duplications of the A. tumefaciens strain Chry5 6b gene promoter. These deletions and duplications of the 6b gene promoter were fused to the GUS reporter gene and transformed into tobacco (Nicotiana tabacum L cv KY160) to monitor levels and tissue specificity of expression. The -284 bp region upstream of 6b gene translational start site was enough for basal expression and was inducible by auxins, 2,4-D and NAA and to a lesser extent by cytokinin. However, the -438 bp fragment showed higher auxin inducibility. The auxin inducibility was increased by duplication of the upstream region fragments of the promoter. GUS expression was mostly confined to the vascular tissue and the meristem region.