Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions.
Plants synthesize the osmoprotectant glycine betaine (GB) via choline-->betaine aldehyde-->glycine betaine. Two enzymes are involved in the pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spinacia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42 degrees C), as determined by immunoblot analysis, but did not respond to cold stress (4 degrees C), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents. (+info
Structural analysis of free and enzyme-bound amaranth alpha-amylase inhibitor: classification within the knottin fold superfamily and analysis of its functional flexibility.
The three-dimensional structure of the amaranth alpha-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to alpha-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution. (+info
Variation in the k(cat) of Rubisco in C(3) and C(4) plants and some implications for photosynthetic performance at high and low temperature.
The capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP is a major limitation on the rate of net CO(2) assimilation (A) in C(3) and C(4) plants. The pattern of Rubisco limitation differs between the two photosynthetic types, as shown by comparisons of temperature and CO(2) responses of A and Rubisco activity from C(3) and C(4) species. In C(3) species, Rubisco capacity is the primary limitation on A at light saturation and CO(2) concentrations below the current atmospheric value of 37 Pa, particularly near the temperature optimum. Below 20 degrees C, C(3) photosynthesis at 37 and 68 Pa is often limited by the capacity to regenerate phosphate for photophosphorylation. In C(4) plants, the Rubisco capacity is equivalent to A below 18 degrees C, but exceeds the photosynthetic capacity above 25 degrees C, indicating that Rubisco is an important limitation at cool but not warm temperatures. A comparison of the catalytic efficiency of Rubisco (k(cat) in mol CO(2) mol(-1) Rubisco active sites s(-1)) from 17 C(3) and C(4) plants showed that Rubisco from C(4) species, and C(3) species originating in cool environments, had higher k(cat) than Rubisco from C(3) species originating in warm environments. This indicates that Rubisco evolved to improve performance in the environment that plants normally experience. In C(4) plants, and C(3) species from cool environments, Rubisco often operates near CO(2) saturation, so that increases in k(cat) would enhance A. In warm-habitat C(4) species, Rubisco often operates at CO(2) concentrations below the K(m) for CO(2). Because k(cat) and K(m) vary proportionally, the low k(cat) indicates that Rubisco has been modified in a manner that reduces K(m) and thus increases the affinity for CO(2) in C(3) species from warm climates. (+info
Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe.
The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3'-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT-PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3'-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3'-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611-949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene. (+info
Decoupling of light intensity effects on the growth and development of C3 and C4 weed species through sucrose supplementation.
Light availability has a profound effect on plant growth and development. One of the ways to study the effects of light intensity on plant growth and development without the confounding problem of photosynthate availability is sucrose injection/supplementation. A greenhouse experiment was conducted to evaluate the effects of light levels (0% and 75% shade) and sucrose injection (distilled water or 150 g sucrose l(-1)) on three weed species: redroot pigweed (Amaranthus retroflexus L., C4), lambsquarters (Chenopodium album L., C3) and velvetleaf (Abutilon theophrasti Medic., C3). The average total sucrose uptake was 7.6 and 5.9 g per plant for 0% and 75% shading, respectively, representing 47% of the average total weed dry weight. Plants injected with sucrose had greater dry weights and shoot-to-root ratios under both light levels. In spite of sucrose supplementation the reduction in dry matter due to shading was greater for roots and reproductive structures than vegetative shoot tissues, indicating light level regulation of morphological changes resulting in changed C allocation that are independent of photosynthate availability. Dry weights of plants injected with sucrose under 75% shading were not different from distilled water-injected unshaded plants. However, both sucrose-injected and control plants, regardless of their photosynthetic pathways, underwent similar changes in allocation of dry matter and morphology due to shading, suggesting that these effects are strictly due to light intensity and not related to photosynthate availability. (+info
Bundle sheath diffusive resistance to CO(2) and effectiveness of C(4) photosynthesis and refixation of photorespired CO(2) in a C(4) cycle mutant and wild-type Amaranthus edulis.
A mutant of the NAD-malic enzyme-type C(4) plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO(2) response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C(4) cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO(2) to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r(bs)) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of r(bs) in A. edulis were 70 to 180 m(2) s(-1) mol(-1), increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO(2) to Rubisco in mesophyll cells of C(3) plants. The values of r(bs) in A. edulis are sufficient for C(4) photosynthesis to elevate CO(2) in BS cells and to minimize photorespiration. The calculated CO(2) concentration in BS cells, which is dependent on input of r(bs), was about 2,000 microbar under maximum rates of CO(2) fixation, which is about six times the ambient level of CO(2). High re-assimilation of photorespired CO(2) was demonstrated in both mutant and wild-type plants at limiting CO(2) concentrations, which can be explained by high r(bs). Increasing O(2) from near zero up to ambient levels under low CO(2), resulted in an increase in the gross rate of O(2) evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO(2) in BS cells. (+info
Antigenic and allergenic properties of Amaranthus Spinosus pollen--a commonly growing weed in India.
Amaranthus spinosus (Fam. Amaranthaceae) is an important aeroallergen in India and grows commonly in different parts of the country. In spite of its clinical significance in Type I hypersensitivity disorders, the antigenic and the allergenic properties of the pollen have not been systematically resolved. We investigated antigenic and allergenic properties of 5 pollen samples of Amaranthus spinosus collected from the Delhi area at fortnightly intervals. The protein content did not exhibit statistically significant variability. However, samples collected during the peak flowering season showed higher protein content. Biochemical characterization of samples showed multiple protein fractions by IEF and SDS-PAGE analysis. Samples collected during peak season showed a slightly higher number of bands (22) in the mw range of 14-70 kD. Seven protein fractions of 70, 66, 60, 50, 40, 30 and 14 kD were observed to have IgE binding capabilities and 9 were treated as allergenic. The observations will be helpful in standardizing pollen antigens for diagnosis and immunotherapy in India. (+info
Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants.
Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 184.108.40.206) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C(4) species), compared to the pattern in Pisum sativum (a C(3) plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C(4)) was 45 degrees C compared to 30 degrees C in P. sativum (C(3)). A. hypochondriacus (C(4)) lost nearly 70% of PEPC activity on exposure to a low temperature of 15 degrees C, compared to only about a 35% loss in the case of P. sativum (C(3)). Thus, the C(4) enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C(3) species. As the temperature was raised from 15 degrees C to 50 degrees C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C(4) plants (45%) was more than that in C(3) species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C(4) plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C(4)) and P. sativum (C(3)) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C(3) and C(4) plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C(4)-PEPC. (+info