Michael J. Muhitch

Transgenic expression of the TRI101 or PDR5 gene increases resistance of tobacco to the phytotoxic effects of the trichothecene 4,15-diacetoxyscirpenol

Mycotoxins are fungal secondary compounds that are toxic to vertebrates. Their presence in food and feeds, as the result of fungal disease in crops, can present a danger to animal or human health. Many mycotoxins have also been shown to be phytotoxic and in some cases, such as with trichothecenes produced by the wheat head blight fungus Fusarium graminearum, mycotoxins may act as virulence factors. Antibiotic-producing organisms, including fungi, protect themselves from their own toxins by metabolic alteration of the compound, modification of the target site of action or by exporting the compound to the extracellular space. We have tested the effectiveness of adapting two of these strategies, metabolic alteration and extracellular transport, to protect plant cells from the deleterious effects of the trichothecene 4,15-diacetoxyscirpenol (DAS). Tobacco plants were transformed with either the Saccharomyces cerevisiae gene PDR5, which encodes a multi-drug transporter, or with the Fusarium sporotrichioides gene TRI101, which encodes a trichothecene 3-O-acetyltransferase. Both genes conferred significant increased tolerance to DAS as measured by a sensitive seed germination assay. Expression of PDR5 or TRI101 in a seed-specific manner in crop plants such as wheat could lower the incidence of head blight as well as reduce mycotoxin levels within the seed.

Effects of expressing E. coli threonine synthase in tobacco (Nicotiana tabacum L.) suspension culture cells on free amino acid levels, aspartate pathway enzyme activities and uptake of aspartate into the cells

Summary The amino acids threonine and methionine are derived from aspartate in a multibranched biosynthetic pathway. In higher plants, the branchpoint enzymes, threonine synthase (TS; EC 4.2.99.2) and cystathionine γ-synthase (CγS; EC 4.2.99.9), which lead to threonine/isoleucine and methionine synthesis, respectively, compete against each other for pathway intermediates. In order to better understand the regulation and interplay between these competing pathways, the feedback-insensitive E. coli TS was constitutively expressed in tobacco ( Nicotiana tabacum L.) suspension cultured cells via Agrobacterium -mediated transformation. Expression of E. coli TS in tobacco cells resulted in a 7-fold increase in total TS activity and a 5.7-fold increase in free threonine levels. CγS activity increased 3.5 fold, apparendy to compensate for the heightened competition for the common pathway intermediate homoserine phosphate. Homoserine dehydrogenase and threonine-sensitive aspartate kinase activities were increased by almost 2 fold. Free aspartate was decreased by 55 % in the transformed cells, while free lysine and isoleucine levels were not significantly changed, indicating that threonine does not regulate its own synthesis by inhibiting an enzyme early in the pathway. Transformed cells had a markedly reduced ability to take up 14 [C] aspartate from the medium, suggesting that threonine may regulate its synthesis in vivo in part by limiting aspartate availability after Met, Lys, and lie pools are filled.

The putative glutamate receptor 3.2 from Arabidopsis thaliana (AtGLR3.2) is an integral membrane peptide that accumulates in rapidly growing tissues and persists in vascular-associated tissues

Abstract The accumulation and localization of the transcript and peptide corresponding to the gene that encodes the putative glutamate receptor isoenzyme 3.2 in Arabidopsis thaliana (AtGLR3.2) is reported. Polyclonal antibodies, raised to the C-terminal region of AtGLR3.2, were used to determine that the putative plant glutamate receptor is an integral membrane protein with an apparent molecular weight of 111±1 kDa. RNA blot analysis revealed temporal accumulation of the AtGLR3.2 transcript in developing seedlings, results that were confirmed by polymerase chain reaction with reverse transcriptase-polymerase chain reaction (RT-PCR). Accumulation of the AtGLR3.2 transcript was highest in rapidly dividing tissues. Immunoblot analysis established that the presence of the AtGLR3.2 peptide mirrored, in most cases, the accumulation of the AtGLR3.2 transcript and suggests that AtGLR3.2 peptide accumulation is controlled in part by gene expression or RNA turnover. Affinity purified antibodies were used to localize the AtGLR3.2 protein in thin tissue sections. Immunohistochemical staining was intense and generalized in the rapidly dividing tissues of the developing floral buds, but mostly confined to the vascular tissue of the more mature hypocotyl, leaf and floral shoot tissues. Localization of the AtGLR3.2 protein to the rapidly growing tissues and vascular tissues is consistent with its proposed role in the translocation of calcium.

Isolation of a promoter sequence from the glutamine synthetase1–2 gene capable of conferring tissue-specific gene expression in transgenic maize

Abstract Glutamine synthetase 1–2 (GS 1–2 ), one of the six glutamine synthetase (GS) genes found in the maize ( Zea mays L.) genome, encodes a cytoplasm-localized GS isozyme (GS p1 ) that is specifically expressed in the basal maternal tissues of the developing kernel. This gene has now been isolated, and its 5′ upstream regulatory region has been sequenced and then used to drive reporter gene expression in stable maize transformants. Expression of the GS 1–2 promoter/GUS heterologous gene resulted in the predicted tissue-specific expression in the basal maternal seed tissues, including the surrounding pericarp. Gene expression within the pedicel parenchyma that subtends the basal endosperm transfer cells and embryo was particularly strong. In contrast, GUS staining was absent in the endosperm and embryo, as well as in leaves or roots. Strong staining was also observed in the basal tissues of developing anthers, which are anatomically similar to the pedicel, and in mature pollen. Silks and husks also stained weakly. A possible explanation for ecotopic expression of GS 1–2 /GUS in pollen is presented. The GS 1–2 expression pattern within the kernel is consistent with the GS p1 isozymes recognized role in nitrogen metabolism during grain fill.

Distribution of the glutamine synthetase isozyme GSp1 in maize (Zea mays).

In maize (Zea mays L.), GSp1, the predominant GS isozyme of the developing kernel, is abundant in the pedicel and pericarp, but absent from the endosperm and embryo. Determinations of GSp1 tissue distribution in vegetative tissues have been limited thus far to root and leaves, where the isozyme is absent. However, the promoter from the gene encoding GSp1 has been shown to drive reporter gene expression not only in the maternal seed-associated tissues in transgenic maize plants, but also in the anthers, husks and pollen (Muhitch et al. 2002, Plant Sci 163: 865-872). Here we report chromatographic evidence that GSp1 resides in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs and stalks of maize plants, but not in mature, shedding pollen grains. RNA blot analysis confirmed these biochemical data. In stalks, GSp1 increased in the later stages of ear development, suggesting that it plays a role in nitrogen remobilization during grain fill.

In vitro metabolism of L-aspartate by maize kernels

Abstract The metabolic fate of l -[14C]aspartate in isolated developing maize (Zea mays) kernels was studied using pulse-chase methods. At the beginning of the chase period, all radioactivity was recovered in the basic (amino acid-containing) fraction of pedicel extracts. Radioactivity recovered in the acid (organic acid-containing) fraction increased steadily from 0 to 24% over the following two hours. Only minor amounts of radioactivity were recovered in the neutral (sugar-containing) fraction over this same period. Alanine was transiently labelled early in the chase period, while glutamine accounted for 15% of the radioactivity recovered in the basic fraction after two hours. The evolution of 14CO2 from [14C]aspartate and the accumulation of aspartate-derived 14C in glutamine, glutamate and alanine were inhibited by the aminotransferase inhibitor aminooxyacetic acid. Seventy-one to 76% of the 14C supplied to the pedicel as [14C]aspartate appeared in the endosperm basic fraction, mainly as glutamine plus glutamate. Direct uptake of glutamine by the endosperm of kernels with pedicels removed was inhibited by 2,4-dinitrophenol and by p-chloromercuribenzenesulphonic acid, suggesting active transport of glutamine by the basal endosperm transfer cells. The role of pedicel amino acid metabolism in maize kernel nitrogen assimilation is discussed.

Excised Tassel-Seed Tunicate (Ts-5 Tu) Kernels as a Model In vitro System for Studying Amino Acid Metabolism in Developing Maize Seeds

Summary Previous studies suggest that nitrogenous transport compounds may be metabolized in the pedicel (basal maternal tissue) of the developing maize ( Zea mays L.) kernel. In this report, excised tassel-seed tunicate ( Ts-5 Tu ) kernels were tested as a model in vitro kernel system to study the metabolic fate of [ l4 C]aspartate within the pedicel and the endosperm. Glume-covered kernels of Ts-5 Tu maize are born on individual elongated stalks and can be removed from the parent plant without damage to the basal kernel tissues. Radiolabeled aspartate supplied to excised Ts-5 Tu kernels in a 30-min pulse was rapidly metabolized within the pedicel, with 60 % of the 14 C recovered in the acidic (organic-acid-containing) fraction at the beginning of the chase period. By 1 h into the chase period, both glutamine and glutamate were more heavily labeled than aspartate. In the endosperm, 60 % of the ethanol-soluble, aspartate-derived 14 C was recovered in the basic (amino acid-containing) fraction, 35% in the acidic fraction, and the remainder in the neutral (sugar-containing) fraction. Radioactivity in glutamate plus glutamine accounted for 70% of the 14 C contained in the basic fraction of the endosperm. Inclusion of 2 mM methionine sulfoximine, an irreversible inhibitor of glutamine synthetase (GS), had little effect on pedicel aspartate catabolism but did inhibit incorporation of aspartate-derived 14 C into glutamine in the pedicel, resulting in more 14 C being taken up by the endosperm as organic acids and less being incorporated into the prolamin fraction. It was concluded that the results obtained using Ts-5 Tu kernels incubated in vitro reflect more closely what occurs in the intact plant than those obtained with cob-borne kernels. The role of pedicel-amino-acid metabolism in maize-kernel nitrogen assimilation is also discussed.

Influence of Nitrogen Source on the Growth, Prolamin Content, and Glutamine Synthetase Isozyme Profiles of Endosperm-Derived Suspension Cultures of Maize

Summary In order to assess the suitability of endosperm-derived suspension cultures (EDSC) as a model system for nitrogen metabolism in intact maize kernel endosperm, the isozym complements of glutamine synthetase (GS), a key enzyme of nitrogen metabolism in higher plants, were compared from both tissue sources. Anion exchange profiles revealed five GS isozymes from intact endosperm. These same isozymes were also found in EDSC, although in very different relative proportions, along with a sixth isozyme with elution characterestics corresponding to the chloroplast GS from maize leaves. Isoelectric focusing under denaturing conditions followed by immunoblotting revealed the presence of five GS subunits from intact endosperm changed strikingly with development. Developmental changes in the isozyme patterns were less dramatic in the EDSC. Culturing the EDSC on alternative nitrogen sources changed the relative proportions of isozymes and affected growth and zein accumulation. It was concluded that while the EDSC do resemble intact endosperm in that they express the same GS subunits, the differences in the relative proportions of those subunits and the corresponding isozymes and differences in developmental changes make extrapolations of results obtained from studies of nitrogen metabolism using the endosperm suspension cultures to the intact endosperm inappropriate.