Influence of Hook Position on Phototropic and Gravitropic Curvature by Etiolated Hypocotyls of Arabidopsis thaliana

Phototropic and gravitropic curvature by hypocotyls of Arabidopsis thaliana is minimal when the side of the hook with the cotyledons attached is positioned toward the direction of tropistic curvature, and maximal when that side of the hook is positioned away from the direction of tropistic curvature. Based on these data, it is proposed that the position of the hook with attached cotyledons affects curvature and not stimulus perception. A randomly oriented population of plants exhibited considerable heterogeneity in tropistic curvature. This heterogeneity arises at least in part from the dependence of curvature on the position of the hook.     

Circumnutations of Arabidopsis thaliana Seedlings

The hypocotyls of Arabidopsis thaliana seedlings exhibit circumnutations with different frequency ranges. Using a picture-analysis system, two types of oscillations were distinguished, short period (SPN) and long period nutations (LPN). The period of the SPNs is between 20 and 60 min, decreases with increasing temperature (between 20° and 30°C; Q 10 of 2.0) and increases with age. The SPNs changed frequently between circular and pendular movements. The circumnutation usually occurred clockwise, but the direction could change. For SPNs to occur, a minimum growth rate of 0.05 mm/h was required. The period of the LPNs ranged from 1 to 8 h. Peaks occurred around 95 and 200 min. The movements of the LPNs are mostly counterclockwise. The oscillation could change from circular to elliptic or pendular. In contrast to SPNs, LPNs were found also at low growth rates. Under red light and in the strain Landsberg erecta the circumnutations were especially slow. Short and long period oscillations can occur simultaneously or separately in different plants. In cases of simultaneous occurrence the quotient between the period lengths of the LPN and SPN was not constant. The number of occurrences of these frequencies depended on the strain and on external factors such as temperature and light. In continuous bright white light phases of circumnutations alternated with quiescent periods. This behaviour was circadian and correlated with growth bursts.     

Circumnutations of Arabidopsis thaliana Seedlings

The hypocotyls of Arabidopsis thaliana seedlings exhibit circumnutations with different frequency ranges. Using a picture-analysis system, two types of oscillations were distinguished, short period (SPN) and long period nutations (LPN). The period of the SPNs is between 20 and 60 min, decreases with increasing temperature (between 20° and 30°C; Q 10 of 2.0) and increases with age. The SPNs changed frequently between circular and pendular movements. The circumnutation usually occurred clockwise, but the direction could change. For SPNs to occur, a minimum growth rate of 0.05 mm/h was required. The period of the LPNs ranged from 1 to 8 h. Peaks occurred around 95 and 200 min. The movements of the LPNs are mostly counterclockwise. The oscillation could change from circular to elliptic or pendular. In contrast to SPNs, LPNs were found also at low growth rates. Under red light and in the strain Landsberg erecta the circumnutations were especially slow. Short and long period oscillations can occur simultaneously or separately in different plants. In cases of simultaneous occurrence the quotient between the period lengths of the LPN and SPN was not constant. The number of occurrences of these frequencies depended on the strain and on external factors such as temperature and light. In continuous bright white light phases of circumnutations alternated with quiescent periods. This behaviour was circadian and correlated with growth bursts.     

Mutants of Arabidopsis thaliana with Decreased Amplitude in Their Phototropic Response

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in `first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.     

Growth Distribution during Phototropism of Arabidopsis thaliana Seedlings

The elongation rates of two opposite sides of hypocotyls of Arabidopsis thaliana seedlings were measured during phototropism by using an infrared imaging system. In first positive phototropism, second positive phototropism, and red light-enhanced first positive phototropism, curvature toward the light source was the result of an increase in the rate of elongation of the shaded side and a decrease in the rate of elongation of the lighted side of the seedlings. The phase of straightening that followed maximum curvature resulted from a decrease in the elongation rate of the shaded side and an increase in the elongation rate of the lighted side. These data for the three types of blue light-induced phototropism tested in this study and for the phase of straightening are all clearly consistent with the growth rate changes predicted by the Cholodny-Went theory.     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花(英文)

AtLH基因是BcpLH基因在拟南芥(Arabidopsis thealiana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域。在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关。为研究AtLH基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥。与野生型比较而言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量表达的植株。这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腋处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强;转基因植株对激素的敏感性改变,IAA刺激根生长的作用增强,ABA抑制根生长的作用减弱。由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲。     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花

AtLH基因是BcpLH基因在拟南芥(Arapsis thaliana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域.在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关.为研究AtLH的基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥.与野生型比较页言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量的植株.这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腑处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强,转基因植株对激素的敏感性改变,IAA剌激根生长的作用增强,ABA抑制根生长的作用减弱.由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲.     

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.     

Light-Stimulated Apical Hook Opening in Wild-Type Arabidopsis thaliana Seedlings

Apical hook opening and cotyledon unfolding are characteristic responses that occur during deetiolation of dicotyledonous seedlings. Light-stimulated apical hook opening and cotyledon unfolding in etiolated Arabidopsis thaliana seedlings appears to involve the activities of multiple photosensory systems. Red, far-red, and blue light are all effective in stimulating these responses in Arabidopsis. Stimulation of hook opening by red light and low fluence blue light is inductive, far-red reversible, and exhibits reciprocity, as is characteristic of many low fluence-dependent phytochrome-mediated responses. Far-red and high-fluence blue light appear to stimulate hook opening and cotyledon unfolding through high-irradiance-response systems during long-term light treatments. Although a phytochrome high-irradiance-response system presumably mediates the responses in far-red light, the responses to high-fluence blue light may be mediated by a blue light-specific photosensory system.     

The G4 Gene of Arabidopsis thaliana Encodes a Chlorophyll Synthase of Etiolated Plants

The gene coding for a putative chlorophyll synthase gene (C4) from Arabidopsis thaliana was amplified by the polymerase chain reaction and cloned into the expression vector pQE- 31. Lysates of bacteria (E.coli) that had been transformed with this construct were used for in vitro enzymatic assays. The chlorophyll synthase catalyzed esterification of chlorophyllides a and b at the same rate but preferred geranylgeranyl-PP over phytyl-PP. This corresponds to the enzyme specificity previously described for etiolated plants and differed from that of green plants.     

Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥(Arabidopsis thaliana)幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明, 在扩增的662条带中, 对照和2个处理及其第2代间完全一致的带型有598条; 发生变化的带型有64条, 其中能遗传给第2代的有48条, 占变异条带的75%。与对照相比, 经超低温保存的样品新产生的甲基化位点有14个, 而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条, 差异条带有38条, 占5.7%, 而对照与未冷冻处理组的差异率是7.45%, 对照与冷冻处理组之间的差异率是6.63%。可见, 拟南芥在超低温保存中, 无论是经液氮冷冻还是未经冷冻处理, 对材料的甲基化状态均有影响, 而这种甲基化变化大部分是可以遗传的。     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥（Arabidopsis thaliana）幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明,在扩增的662条带中,对照和2个处理及其第2代间完全一致的带型有598条：发生变化的带型有64条,其中能遗传给第2代的有48条,占变异条带的75％。与对照相比,经超低温保存的样品新产生的甲基化位点有14个,而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条,差异条带有38条,占5.7％,而对照与未冷冻处理组的差异率是7．45％,对照与冷冻处理组之间的差异率是6。63％。可见,拟南芥在超低温保存中,无论是经液氮冷冻还是未经冷冻处理,对材料的甲基化状态均有影响,而这种甲基化变化大部分是可以遗传的。     

Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana

Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana , three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.     

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago ¹ and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) ^2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) ^2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 10⁶-10⁷ protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.Download video file.^{(131M, mp4)}     

Necessity of Superoxide Production for Development of Etiolated Wheat Seedlings

It was found that production of superoxide (O₂ ^{– ·}) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O₂ ^{– ·} production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O₂ ^{– ·} production was clearly observed in the period from two to four days of seedling development, then the rate of O₂ ^{– ·} production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O₂ ^{– ·} production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O₂ ^{– ·} production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O₂ ^{– ·} amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O₂ ^{– ·} controls plant growth by cell elongation at the early stages of seedling development but later O₂ ^{– ·} controls (induces) apoptotic DNA fragmentation and protein disintegration.