Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.     

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.     

G-proteins in etiolated Avena seedlings. Possible phytochrome regulation

The molecular mechanism of light signal transduction in plants mediated by the photosensor phytochrome is not well understood. The possibility that phytochrome initiates the signal transduction chain by modulating a G-protein-like receptor is examined in the present work. Etiolated Avena seedlings contain G-proteins as examined in terms of the binding of GTP as well as by cross-reaction with mammalian G-protein antibodies. The binding of GTP was regulated in vivo by red/far-red light. The possible involvement of G-proteins in the phytochrome-mediated signal transduction in etiolated Avena seedlings has been implicated from the study of the light regulated expression of the Cab and phy genes.     

Pigments formed in etiolated sunflower seedlings

Etiolated plants must be irradiated before chlorophyll can form in them. Chlorophyll develops most readily in young irradiated plants. More carotene develops in seedlings grown from young seeds than in seedlings grown from older ones. There may be a relationship between the viability of seeds and the potential power to produce carotene. Carotinoid-pigment formation precedes chlorophyll formation. Probably some necessary substance is translocated in the plant for the formation of pigment. Carotene and xanthophyll may be precursors of chlorophyll. They probably form a respiratory mechanism for the plant. Carotene may be a precursor of auxin.     

Ubiquitin C-terminal hydrolases 1 and 2 affect shoot architecture in Arabidopsis

Ubiquitin C-terminal hydrolases (UCHs) are a subset of de-ubiquitinating proteases that release covalently linked ubiquitin (Ub), and as such play essential roles in recycling Ub and reversing the action of Ub conjugation. We show here that two related Arabidopsis UCHs, UCH1, and UCH2, are important for shoot development. The UCH1 and 2 genes are ubiquitously expressed, with the corresponding proteins present in both the cytoplasm and nucleus. Unlike their animal and fungal counterparts, we found no evidence that the Arabidopsis UCH1 and 2 proteins stably associate with the 26S proteasome. Altering the levels of UCH1 and 2 has substantial effects on Arabidopsis shoot development, especially with respect to inflorescence architecture, with over-expression and double mutants enhancing and suppressing the outgrowth of cauline branches, respectively. Neither UCH1-over-expressing nor uch1-1 uch2-1 plants have detectably altered sensitivity to cytokinins or auxins individually, but exhibit an altered sensitivity to the ratio of the two hormones. UCH1-over-expressing plants show dramatically enhanced phenotypes when combined with auxin-insensitive mutants axr1-3 and axr2-1, suggesting that one or more aspects of auxin signaling are affected by this enzyme pair. Previous studies revealed that the ubiquitination and degradation of the AUX/IAA family of repressors is a key step in auxin signaling. Here, we show that turnover of a reporter fused to a representative AUX/IAA protein AXR3 is faster in the uch1-1 uch2-1 double mutant but slower in the UCH1 over-expression backgrounds. Taken together, our results indicate that de-ubiquitination helps to modify plant shoot architecture, possibly via its ability to directly or indirectly protect upstream target proteins involved in auxin/cytokinin signaling from Ub-mediated degradation.     

Tetrapyrrole biosynthesis in greening etiolated barley seedlings

Allyl isopropylacetamide (AIA) does not stimulate porphyrin biosynthesis in greening barley; AIA inhibits the synthesis of 5-aminolaevulinate (ALA) in plants and does not overcome the repression of ALA-synthetase. This indicates that the ALA synthesis system of green plants is regulated differently from ALA synthetase of mammalian systems. Laevulinic acid (LA) inhibited the biosynthesis of tetrapyrrole pigments in greening barley and diminished the insertion of ⁵⁵Fe into extractable protohaem, confirming that haem was synthesized at a time of little net increase in protohaem. ALA feeding increased iron incorporation into protohaem without increasing either extractable protohaem or cytochromes b and f. Since ALA feeding greatly increased the protochlorophyllide content of darkgrown plants and subsequent chlorophyll levels in the light, the regulation of haem pigment synthesis in plants occurs after protoporphyrin and protohaem synthesis and is likely to involve the turnover of protohaem produced in excess of haem protein requirements.     

Superoxide dismutase in wounded etiolated pea seedlings

The activities of three related enzymes, superoxide dismutase, peroxidase and catalase, were followed in decapitated (wounded) etiolated pea stem tissue. Of the three enzyme activities, only that of superoxide dismutase showed a significant loss and recovery pattern within 1 hr. The change in enzymatic activity appears to result from protein loss and recovery.     

拟南芥PKS2与CAM4蛋白互作研究

植物蓝光受体向光素(phototropin,PHOT)介导许多生理反应,现已从拟南芥中分离了其下游的一些信号转导组分。前期研究表明,拟南芥光敏色素底物PKS家族成员PKS1与部分Ca2+结合蛋白钙调素(calmodulin,CAM)成员互作,参与PHOT2介导的强蓝光诱导下胚轴向光反应。旨在探讨PKS2和CAM4之间的互作关系,首先用RT-PCR技术得到PKS2和CAM4的c DNA全长序列。通过酵母双杂交和双分子荧光互补技术,从体外与体内证实PKS2和CAM4能相互作用。此结果进一步丰富了PKS家族与CAM之间的联系,为深入解析PHOT功能研究奠定基础。     

Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings.

Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein.h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein.h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

UV-B response of green and etiolated barley seedlings

7-d-old etiolated and green barley seedlings (Hordeum vulgare L. cv. Alfa) were irradiated with UV-B for 30 min and then kept for 24 h in light or darkness. Chlorophyll (Chl) synthesis was inhibited by about 30 % as a result of UV-B irradiation, but there were no significant changes in photochemical activity measured by variable to maximum fluorescence ratio (F_v/F_m), quantum yield (Φ_PS2) and oxygen evolution rate. Electron transport of etiolated seedlings was similar to that of green ones, nevertheless, the Chl content was more then 2-fold lower. Ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits were diminished as a result of UV-B irradiation in etiolated and green plants, especially in those kept in the darkness. Catalase activity decreased and total superoxide dismutase activity increased in green and etiolated plants following UV-B treatment. When benzidine was used as a substrate, an isoform located between guaiacol peroxidases 2 and 3 (guaiacol peroxidase X) appeared, which was specific for UV-B treatment. As a result of irradiation, the contents of UV-B absorbing and UV-B induced compounds increased in green seedlings but not in etiolated seedlings.     

UDPG:Sterol glucosyltransferase in etiolated pea seedlings

Sterol UDPglucose glucosyltransferase was located predominantly in the axis tissue of etiolated pea seedlings. During the first 11 days of growth the activity reached a peak in the axis tissue after seven days. Centrifugation of tissue homogenates showed the cell fraction sedimenting between 13000 nd 25000 g to have the highest specific activity and also the bulk of the total activity. Sitosterol is the major free sterol of this fraction and cholesterol is a trace component. The composition of the aglycones of the isolated steryl glycosides shows cholesterol to be the major sterol. Although exhibiting no metal ion requirement, the enzyme is stimulated by Ca²⁺ and Mg²⁺, partially inhibited by EDTA and EGTA and completely inhibited by Zn²⁺. The membranous nature of the enzyme is manifested by its stimulation by the addition of phosphatidyl -ethanolamine, -choline and -serine. After brief treatment with phospholipases A, C and D, enzyme activity is partially lost. After phospholipase A treatment the activity may be completely restored by the addition of phosphatidyl ethanolamine but phosphatidyl-choline and -serine are without effect. After phospholipase C and D treatment, each phospholipid brings about a partial recovery of activity but phosphatidyl ethanolamine is again superior.     

Apoplastic domains and sub-domains in the shoots of etiolated corn seedlings

Light Green, an apoplastic probe, was applied to the cut mesocotyl base or to the cut coleoptile apex of etiolated seedlings of Zea mays L. cv. Silver Queen. Probe transport was measured and its tissue distribution determined. In the mesocotyl, there is an apoplastic barrier between cortex and stele. This barrier creates two apoplastic domains which are non-communicating. A kinetic barrier exists between the apoplast of the mesocotyl stele and that of the coleoptile. This kinetic barrier is not absolute and there is limited communication between the apoplasts of the two regions. This kinetic barrier effectively creates two sub-domains. In the coleoptile, there is communication between the apoplast of the vascular strands and that of the surrounding cortical tissue. No apoplastic communication was observed between the coleoptile cortex and the mesocotyl cortex. Thus, the apoplastic space of the coleoptile cortex is a sub-domain of the integrated coleoptile domain and is separate from that of the apoplastic domain of the mesocotyl cortex.     

Light regulates symplastic communication in etiolated corn seedlings

The effect of light on symplastic communication in dark grown maize seedlings was tested using the symplastic probe carboxyfluorescein. Prior white-light irradiation stimulated longitudinal transport of carboxyfluorescein in the mesocotyl stele of dark-grown seedlings. The stimulation was multiphasic with a positive followed by a negative phase. Lateral transport from the stele into the mesocotyl cortex was inhibited by the prior white-light irradiation. The inhibitory effect of prior white-light irradiation on lateral transport was completely photo-modulatable by terminal farred and far-red/red irradiations, suggesting the involvement of phytochrome. The stimulatory effect of the prior white irradiation observed for the stele, however, was not photoreversible. It is suggested that environmental factors, such as light, might modulate growth and development, in part, by modulating symplastic cell to cell communication and thus the distribution of nutrients and growth regulators.     

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.