Variations in sterol composition in etiolated mung bean seedlings

The sterol fractions of different morphological and physiological parts of etiolated Mung bean seedlings have been studied. Young growing tissues conta     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Proteomic responses in Arabidopsis thaliana seedlings treated with ethylene

Ethylene (ET) is a volatile hormone that modulates fruit ripening, plant growth, development and stress responses. Key components of the ET-signaling pathway identified by genetic dissection in Arabidopsis thaliana include five ET receptors, the negative regulator CTR1 and the positive regulator EIN2, all of which localize to the endoplasmic reticulum. Mechanisms of signaling among these proteins are still unresolved and targets of ET responses are not fully known. So, we used mass spectrometry to identify proteins in microsomal membrane preparations from etiolated A. thaliana seedlings maintained in ambient air or treated with ET for 3 h. We compared 3814 proteins from ET-exposed seedlings and controls and identified 304 proteins with significant accumulation changes. The proteins with increased accumulation were involved in ET biosynthesis, cell morphogenesis, oxidative stress and vesicle secretion while those with decreased accumulation were ribosomal proteins and proteins positively regulated by brassinosteroid, another hormone involved in cell elongation. Several proteins, including EIN2, appeared to be differentially phosphorylated upon ET treatment, which suggests that the activity or stability of these proteins may be controlled by phosphorylation. TUA3, a component of microtubules that contributes to cellular morphological change, exhibited both increased accumulation and differential phosphorylation upon ET treatment. To verify the role of TUA3 in the ET response, tua3 mutants were evaluated. Mutant seedlings had altered ET-associated growth movements. The data indicate that ET perception leads to rapid proteomic change and that these changes are an important part of signaling and development. The data serve as a foundation for exploring ET signaling through systems biology.     

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.     

Alterations of NADPH:protochlorophyllide oxidoreductase quantity and lipid composition in etiolated barley seedlings infected by Barley stripe mosaic virus (BSMV)

To understand the phenomenon by which infection of seed-transmitted Barley stripe mosaic virus (BSMV) alters membrane structures and inhibits protochlorophyllide biosynthesis of dark-grown barley ( Hordeum vulgare L.) plants, we analysed the presence of NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) and the galactolipid content and fatty acid composition. The amount of POR in etioplasts of infected leaves, compared with non-infected leaves, was reduced, as measured by immunoelectron microscopy and Western blot. These results are in agreement with the previously described reduction of the ratio of the photoactive 650 nm to non-photoactive 630 nm absorbing protochlorophyllide forms ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The galactolipid content was lower in infected leaves. Monogalactosyl-diacylglycerol (MGDG) content was reduced to 40% and digalactosyl-diacylglycerol to 55% of control plants on a fresh weight basis. In infected plants, the proportion of linolenic acid decreased in both galactolipids. The lower amount of highly unsaturated fatty acids and the reduced abundance of MGDG correlated well with the previously detected reduction in the membrane ratio of prolamellar body (PLB) to prothylakoid ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The reduced amount of POR and the above described alterations in the lipid composition resulted in a disturbed structure of PLBs. As a consequence, pigment synthesis and the greening process were inhibited in infected cells, in turn explaining the appearance of chlorotic stripes of BSMV-infected barley leaves. Our results show that BSMV infection can be detected at a very early stage of leaf development.     

Catalytic function of a novel protein protochlorophyllide oxidoreductase C of Arabidopsis thaliana

In Arabidopsis thaliana Por C has been identified only on sequence homology to that of por A and por B. To demonstrate its catalytic function Arabidopsis thaliana protochlorophyllide oxidoreductase C gene (por c) that codes for the mature part of POR C protein having 335 amino acids was expressed in Escherchia coli cells. The POR C enzyme in the presence of NADPH and protochlorophyllide when incubated in dark formed a ternary complex. When it was excited at 433 nm, it had a fluorescence emission peak at 636 nm. After illumination with actinic cool white fluorescent light, a peak at 673 nm due to chlorophyllide gradually increased with concomitant decrease of 636 nm emission, demonstrating the gradual phototransformation of protochlorophyllide to chlorophyllide. The significance of differential por gene expression in light and dark among different species is discussed.     

The Lhcb protein and xanthophyll composition of the light harvesting antenna controls the DeltapH-dependency of non-photochemical quenching in Arabidopsis thaliana

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the DeltapH-dependent component of NPQ (qE) component of non-photochemical quenching is controlled allosterically by the xanthophyll cycle has been tested using Arabidopsis mutants with different xanthophyll content and composition of Lhcb proteins. The titration curves of qE against DeltapH were different in chloroplasts containing zeaxanthin or violaxanthin, proving their roles as allosteric activator and inhibitor, respectively. The curves differed in mutants deficient in lutein and specific Lhcb proteins. The results show that qE is determined by xanthophyll occupancy and the structural interactions within the antenna that govern allostericity.     

Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings

Etiolated Arabidopsis thaliana seedlings, lacking a functional prephenate dehydratase1 gene (PD1), also lack the ability to synthesize phenylalanine (Phe) and, as a consequence, phenylpropanoid pigments. We find that low doses of ultraviolet (UV)-C (254 nm) are lethal and low doses of UV-B cause severe damage to etiolated pd1 mutants, but not to wild-type (wt) seedlings. Furthermore, exposure to UV-C is lethal to etiolated gcr1 (encoding a putative G protein-coupled receptor in Arabidopsis) mutants and gpa1 (encoding the sole G protein alpha subunit in Arabidopsis) mutants. Addition of Phe to growth media restores wt levels of UV resistance to pd1 mutants. The data indicate that the Arabidopsis G protein-signalling pathway is critical to providing protection from UV, and does so via the activation of PD1, resulting in the synthesis of Phe. Cotyledons of etiolated pd1 mutants have proplastids (compared with etioplasts in wt), less cuticular wax and fewer long-chain fatty acids. Phe-derived pigments do not collect in the epidermal cells of pd1 mutants when seedlings are treated with UV, particularly at the cotyledon tip. Addition of Phe to the growth media restores a wt phenotype to pd1 mutants.     

Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC-YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.     

The xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana

Arabidopsis plants overexpressing beta-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Supercooling capacity of seeds and seedlings in Arabidopsis thaliana.

The influence of the water content of seeds and seedlings of Arabidopsis thaliana (Ecotype Columbia:2) on their supercooling capacity was investigated. Equilibration of the seeds to various air relative humidities resulted in final moisture contents ranging from 8 to 82% (dry weight basis). No supercooling point could be detected when the water content remained below 32.5%, and in seeds at just above this moisture level ice crystals started to form at -26 degrees C. However, cooling partly affected the germination of seeds down to a water content of 26.5%. Upon imbibition, the supercooling point of the seeds remained around -21.6 degrees C and rose sharply to -14.7 degrees C when visible germination started. It remained around -13 degrees C during the following 96 h while the water content of the seedlings increased from 155 to 870%. Hydrated seeds (above 32.5% water content), germinated seeds, and seedlings of Arabidopsis cannot survive being frozen.     

Mutants of Arabidopsis thaliana with altered cell wall polysaccharide composition

To analyze the synthesis, structure and function of the plant cell wall by a genetic approach, 5200 chemically mutagenized Arabidopsis plants were screened for changes in the monosaccharide composition of hydrolyzed cell wall material by gas chromatography of alditol acetates. This screening procedure identified 23 mutant lines representing 11 different loci designated mur1 to mur11 . The mur lines fall into essentially three groups: (1) complete absence of a monosaccharide, (2) significant reduction in the amount of a single monosaccharide, and (3) complex alterations in the relative amounts of several monosaccharides. All mutants in the first category represent alleles of the mur1 locus, and are deficient in the de novo synthesis of fucose. Mutants with reductions in a single monosaccharide have been identified for fucose ( mur2, mur3 ), arabinose ( mur4, mur5, mur6, mur7 ), and rhamnose ( mur8 ). Mutants with complex changes in monosaccharide composition are represented by the mur9 , mur10 and mur11 loci. Most of the mutant lines did not show obvious morphological or physiological alterations; however, lines mur1, mur9 and mur10 co-segregated with reduced vigor or dwarfism of the plants. These results demonstrate the feasibility of identifying plants with altered cell wall compositions via a biochemical screening procedure. The availability of these mutants provides novel opportunities to study the functions of cell wall polysaccharides, gain insight into the biosynthesis of cell wall material, and clone cell wall-related genes.