Genetic dissection of plant cell-wall biosynthesis.

The plant cell wall is a complex structure consisting of a variety of polymers including cellulose, xyloglucan, xylan and polygalacturonan. Biochemical and genetic analysis has made it possible to clone genes encoding cellulose synthases (CesA). A comparison of the predicted protein sequences in the Arabidopsis genome indicates that 30 divergent genes with similarity to CesAs exist. It is possible that these cellulose synthase-like (Csl) proteins do not contribute to cellulose synthesis, but rather to the synthesis of other wall polymers. A major challenge is, therefore, to assign biological function to these genes. In an effort to address this issue we have systematically identified T-DNA or transposon insertions in 17 Arabidopsis Csls. Phenotypic characterization of "knock-out" mutants includes the determination of spectroscopic profile differences in mutant cell walls from wild-type plants by Fourier-transform IR microscopy. A more precise characterization includes cell wall fractionation followed by neutral sugar composition analysis by anionic exchange chromatography.

Visualizing rhizosphere chemistry of legumes with mid-infrared synchrotron radiation.

A bright synchrotron light source operated by the Lawrence Berkeley National Laboratory served as an external source for infrared (IR) microscopy of plant root microcosms. Mid-IR light from synchrotrons is 2-3 orders of magnitude brighter than conventional sources, providing contrast based on the chemical information in the reflected signal at a spatial resolution near the diffraction-limit of 3-10 microm. In an experiment using plant root microcosms fitted with zinc selenide IR-transmissive windows (50 mm x 20 mm x 1 mm), we describe chemical differences and similarities within the root zone of mung bean (Vigna radiata L.), grown with or without phosphorus, and revealed by reflectance spectromicroscopy. Comparative root and root-exudate profiles are described in sand/silt culture over the wavelength range of 2.5 to 16 pm (4.000 to 650 cm(-1) ) in the mid-IR. the spectral region most useful for the analytical identification of small organic molecules. Root epidermal tissue of plants grown with low phosphorus showed a greater lipid contribution and less lignin than nutrient-sufficient plants. In the zone 200 microm from the root axis, control plants were enriched with simple sugars and monomeric lignin precursors. In low-phosphorus plants, the rhizosphere possessed IR signatures from protein and sugars. Individual soil minerals could be easily discriminated from biological material. Synchrotron IR spectromicroscopy, therefore, complements existing root imaging techniques.

Carbon, Nitrogen, and Nutrient Interactions in Beta vulgaris L. as Influenced by Nitrogen Source, NO3- versus NH4+

Sugar beets (Beta vulgaris L. cv F58-554H1) were grown hydroponically in a 16-h light, 8-h dark period (photosynthetic photon flux density of 0.5 mmol m-2 s-1) for 4 weeks from sowing in half-strength Hoagland nutrient solution containing 7.5 mM nitrate. Half of the plants were then transferred to 7.5 mM ammonium N; the rest remained in solution with 7.5 mM nitrate N. Upon transfer from nitrate to ammonium, the total N concentration decreased sharply in the fibrous roots and petiole/midribs and increased substantially in the leaf blades. This was because of the decreased nitrate concentrations in fibrous roots and petioles and a concomitant increase in amino acid/amide-N and protein N in leaf blades. Sugar beets acclimated to ammonium partly by a 2.5-fold increase in glutamine synthase activity in fibrous roots and a 1.7-fold increase in leaf blades. Rapid ammonium assimilation into glutamine consumed carbon skeletons, leading to a depletion of foliar starch, sucrose, and maltose. Ammonium treatment stimulated activities of some glycolytic/Krebs cycle enzymes, e.g. pyruvate dehydrogenase. Nitrate-fed leaf blades contained substantially larger concentrations of osmolytes (i.e. nitrate, cations, and sucrose), which may have contributed to the faster rates of leaf expansion in nitrate-fed compared to ammonium-fed plants.

Nitrogen Source Regulation of Growth and Photosynthesis in Beta vulgaris L.

Sugar beets (Beta vulgaris L. cv F58-554H1) were grown hydroponically in a 16-h light, 8-h dark period at a photosynthetic photon flux density of 0.5 mmol m-2 s-1 for 4 weeks in half-Hoagland culture solution containing only nitrate-nitrogen. Half of the plants were then transferred to half-Hoagland solution with ammonium-nitrogen (7.35mM), while the other half continued on 7.5 mM nitrate. Growth analysis was carried out by sampling the plants at 3-d intervals over a period of 21 d. Compared to plants supplied with nitrate, ammonium initially slowed the growth of shoots more than roots. Ammonium reduced both the area expansion of individual leaves and the relative water content of these leaves, but increased the amount of dry matter/area. The increase in specific leaf weight in ammonium-grown leaves was associated with a doubling of chloroplast volume, as much as a 62% rise in chlorophyll content, and a 4.3-fold higher accumulation of soluble protein. Ammonium nutrition substantially decreased the rate of expansion of photosynthetic (leaf) surface but did not decrease the rate of photosynthesis per area; in fact, net photosynthetic CO2 exchange rates were slightly higher than in nitrate plants, due to the build-up in stromal enzymes of the Calvin cycle, several of which increased in total extractable activity on a leaf area basis, e.g. ribulose-1,5- biphosphate carboxylase oxygenase, sedoheptulose-1,7-biphosphatase. Nitrogen source had no effect on stomatal conductance. Rates of photosynthesis per chlorophyll were decreased slightly in ammonium-grown leaves, possibly due to an increased CO2-diffusion resistance associated with the enlarged chloroplasts.

Effects of phosphorus nutrition on photosynthesis in Glycine max (L.) Merr.

The effects of phosphorus nutrition on various aspects of photosynthetic metabolism have been examined for soybean plants (Glycine max) grown in growth chambers. Orthophosphate was supplied at two levels in 0.5-strength Hoagland's solution. At the end of the 19-d growth period, plants grown at 10 µM KH2PO4 (low-P plants) had undergone a 40% drop in net CO2 exchange (averaged over a 16-h light period), as compared with control plants grown with 200 µM KH2PO4. Low-P resulted in reductions in the initial activities of five, and in the total activities of seven, Calvin-cycle enzymes. Notable exceptions were the initial and total activities of chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) which were increased by 85 and 53%, respectively, by low-P. Low-P decreased leaf 3-phosphoglycerate (PGA) levels most (by 80%), ribulose-1,5-bis-phosphate (RuBP) less (by 47%) while triose-phosphate (TP) was not significantly changed. The results indicate that photosynthetic CO2-fixation in low-P plants was limited more by RuBP regeneration than by ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) activity. Ribulose-1,5-bisphosphate regeneration in low-P plants did not appear to be limited by ATP and-or NADPH supply because ATP/ADP and NADPH/ NADP(+) ratios were increased by 60 and 37%, respectively, by low-P, and because TP/PGA ratios were higher in low-P plants. Low-P may diminish RuBP regeneration, and hence photosynthesis, by reducing Calvin-cycle enzyme activity, in particular, the initial activity of ribulose-5-phosphate kinase (EC 2.7.1.19) (44% reduction), and by enhancing the flux of carbon into starch biosynthesis.