A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize.

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.

Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues.

Metabolic engineering for production of isoflavones in non-legume plants may provide the health benefits of these phytoestrogens from consumption of more widely used grains. In legumes, isoflavones function in both the symbiotic relationship with rhizobial bacteria and the plant defense response. Expression of a soybean isoflavone synthase (IFS) gene in Arabidopsis plants was previously shown to result in the synthesis and accumulation of the isoflavone genistein in leaf and stem tissue (Jung et al., 2000). Here we further investigate the ability of the heterologous IFS enzyme to interact with the endogenous phenylpropanoid pathway, which provides the substrate for IFS, and produces genistein in several plant tissue systems. In tobacco (Nicotiana tabacum) floral tissue that synthesizes anthocyanins, genistein production was increased relative to leaves. Induction of the flavonoid/anthocyanin branch of the phenylpropanoid pathway through UV-B treatment also enhanced genistein production in Arabidopsis. In a monocot cell system, introduced expression of a transcription factor regulating genes of the anthocyanin pathway was effective in conferring the ability to produce genistein in the presence of the IFS gene. Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of IFS resulting in production of the isoflavone daidzein in this system. The genistein produced in tobacco, Arabidopsis, and maize (Zea mays) cells was present in conjugated forms, indicating that endogenous enzymes were capable of recognizing genistein as a substrate. This study provides insight into requirements for metabolic engineering for isoflavone production in non-legume dicot and monocot tissues.

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression.

The Arabidopsis APETALA3 (AP3) and PISTILLATA (PI) proteins are thought to act as transcription factors and are required for specifying floral organ identities. To define the nuclear localization signals within these proteins, we generated translational fusions of the coding regions of AP3 and PI to the bacterial uidA gene that encodes beta-glucuronidase (GUS). Transient transformation assays of either the AP3-GUS or PI-GUS fusion protein alone resulted in cytoplasmic localization of GUS activity. However, coexpression of AP3-GUS with PI, or PI-GUS with AP3, resulted in nuclear localization of GUS activity. Stable transformation with these fusion proteins in Arabidopsis showed similar results. The nuclear colocalization signals in AP3 and PI were mapped to the amino-terminal regions of each protein. These observations suggest that the interaction of the AP3 and PI gene products results in the formation of a protein complex that generates or exposes a colocalization signal required to translocate the resulting complex into the nucleus. The colocalization phenomenon that we have described represents a novel mechanism to coordinate the functions of transcription factors within the nucleus.

C4 isoform of NADP-malate dehydrogenase. cDNA cloning and expression in leaves of C4, C3, and C3-C4 intermediate species of Flaveria.

In C4 plants of the NADP-malic enzyme type, an abundant, mesophyll cell-localized NADP-malate dehydrogenase (MDH) acts to convert oxaloacetate, the initial product of carbon fixation, to malate before it is shuttled to the bundle sheath. Since NADP-MDH has different but important roles in leaves of C3 and C4 plants, we have cloned and characterized a nearly full-length cDNA encoding NADP-MDH from Flaveria trinervia (C4) to permit comparative structure/expression studies within the genus flaveria. The dicot genus Flaveria includes C3-C4 intermediate species, as well as C3 and C4 species. We show that the previously noted differences in NADP-MDH activity levels among C3, C4, and C3-C4 Flaveria species are in part due to interspecific differences in mRNA accumulation. We also show that the NADP-MDH gene appears to be present as a single copy among different Flaveria species, suggesting that a pre-existing gene has been reregulated during the evolution from C3 to C4 plants to accommodate the abundance and localization requirements of the C4 cycle.

A model of transitive choice.

We report a formal model of transitive inference based on protocols from experiments on squirrel monkeys solving the 5-term series problem (McGonigle & Chalmers, 1977, 1992). These studies generate databases featuring transitive choice, task transfer (where at first a significant decrement is observed, and later substantial improvement without explicit training), and, finally, a Symbolic Distance Effect (SDE) based on decision-time data. Using a rule-based (production) system, we first established rule stacks at the group, then at the individual level, on the basis of triadic transfer performance first recorded in the McGonigle and Chalmers (1977) study. The models for each subject then accommodated data from the more intensive, later study with the same subjects (McGonigle & Chalmers, 1992). We found the initial model capable of dealing with all choice and reaction-time phenomena reported thus far, with only small changes in a rule search procedure. In common with an independent assay by McGonigle and Chalmers (1992), our model-based reassessment of decision times indicates that a major source of reaction time variation is item prominence in the rule stack rather than interitem (ordinal) distance per se.

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis.

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O(2)-resistant photosynthesis. The apparent K(m) for H(2)O(2) was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco.

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N(6)-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation.

Attachment as a factor in the protection of Enterobacter cloacae from chlorination.

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.

The selective impact of question form and input mode on the symbolic distance effect in children.

Five experiments are reported on the symbolic distance effect (SDE) and related phenomena with 6- and 9-year-old children. In the first of these, children were asked to judge the relative sizes of animals in verbal and pictorial tests featuring the comparatives "bigger" and "smaller." A perceptual condition with actual objects was included by way of comparison. A Symbolic Distance Effect was obtained for both lexical and pictorial input. Mode differences were also observed. Pictures produced faster responses than words, and congruity effects occurred only in the pictorial condition. Although performance was similar in tests with either comparative, our subsequent experiments on both 6- and 9-year-olds reveal a significant asymmetry in the child's capacity to verify statements of relation as a function of the direction along the (size) continuum implied by the question. However, important differences between age groups also apparent in the data lead us to conclude that the older subjects develop strategies to overcome this asymmetry by translating certain statements of relation into a form more congruent with their natural modes of encoding.

The role of context in the perception of orientation by the rat.

Two experiments are reported which demonstrate the role of contextual factors in the rat's perception of orientation. Designed to eschew criticisms of an earlier study by McGonigle and Jones, the first experiment shows that the rat's perception of linear Gestalten is conditional upon the set of alternative patterns within which the (linear) pattern is embedded. The second experiment shows that the ecological framework of an orientational cue (rather than its position relative to retinocortical coordinates) determines the rat's perception of vertical and horizontal stimuli.

The selective impact of verbally communicated criteria in orientation judgements by young children.

In a series of tests requiring judgements of orientation, the impact of a verbally transmitted equivalence criterion (e.g. "Pick me out the one exactly the same as this one") was compared with one which specified the 'identity' of the target as a 'standing up' or 'lying down' pattern. When a subset of four-year-olds was given the latter instruction, their performance improved dramatically (although all stimuli were lying flat on a horizontal surface) but reverted to a previous (low) level when equivalence instructions were reintroduced. The results suggest that findings from equivalence tests cannot be used to make unambiguous inferences concerning the competence of young children to make identification judgements.

Levels of stimulus processing by the squirrel monkey: relative and absolute judgements compared.

Nine squirrel monkeys were required to select from various sets of stimuli-differing in size or brightness-either in terms of relational criteria or on an absolute stimulus basis. The level of information processing required by each task was assessed by means of stimulus transformation techniques, variations in set size, and by the elimination of the visible context. It was found that some relational judgements make fewer processing demands on the subject than do absolute stimulus judgements; the 'middle' relation, however, appears much more difficult to use than selection of a stimulus on an absolute basis and may be beyond the competence of the squirrel monkey. The results are seen as support for the thesis as advanced by McGonigle and Jones that the criteria of judgement, when varied, change the depth of stimulus processing by monkey as well as man.