Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. (II. Cold Induction of Enzymes in Gibberellin Biosynthesis)

Vernalization of Thlaspi arvense L. results in the alteration of gibberellin (GA) metabolism such that the metabolism and turnover of the GA precursor ent-kaur-16-en-19-oic acid (kaurenoic acid) is dramatically increased. This cold-induced change in GA metabolism is restricted to the shoot tip, the site of perception of cold in this species (J.P. Hazebroek, J.D. Metzger [1990] Plant Physiol 94: 157-165). In the present report additional biochemical information about the nature of this low-temperature-regulated process is provided. The endogenous levels of kaurenoic acid in leaves and shoot tips of plants were estimated by combined gas chromatography-chemical ionization mass spectrometry at various times after 4 weeks of vernalization at 6[deg]C. The endogenous levels in shoot tips declined 10-fold by 2 d after the plants were returned to 21[deg]C; this decline continued such that there was nearly 50-fold less kaurenoic acid by 10 d after the end of vernalization. No effect of vernalization on the endogenous levels of kaurenoic acid in leaves was observed. An in vitro enzyme assay was developed to monitor changes in the ability of tissues to convert kaurenoic acid to ent-7[alpha]-hydroxykaur-16-en-19-oic acid (7-OH kaurenoic acid). The activity of this enzyme rapidly increased in microsomal extracts from shoot tips following the end of vernalization. No thermoinduced increase in activity was observed in leaves. The enzymic oxidation of ent-kaurene to ent-kaurenol was also induced in shoot tips by vernalization. However, this reaction does not appear to be rate limiting for GA biosynthesis, because substantial amounts of kaurenoic acid accumulated in noninduced shoot tips. These results corroborate our hypothesis that the conversion of kaurenoic acid to 7-OH kaurenoic acid is the primary step in GA metabolism regulated by vernalization in Thlaspi shoot tips.     

Gibberellins and Light Regulated Petiole Growth in Thlaspi arvense L

Petiole growth in Thlaspi arvense L. was stimulated when a basic 8 hour photoperiod (4.20 milliwatts per square centimeter) was extended with low intensity light (0.16 milliwatt per square centimeter) from incandescent lamps. The day length extension was effective only when the light contained high proportions of far red light. Exogenous gibberellin A₃ (GA₃) could partially substitute for the promotive effect of the extended photoperiod. Moreover, the GA biosynthesis inhibitor 2-chlorocholine chloride inhibited the increase in petiole growth induced by the extended photoperiod. However, evidence was obtained indicating that gibberellins do not mediate the effect of the extended photoperiod. First, petiole growth was greater in plants receiving both exogenous GA₃ and a day length extension than the sum of the effects of the two treatments alone. Second, petioles were sensitive to exogenous GA₃ only during the early stages of leaf development, whereas mature (but not senescent) leaves continued to respond to an extension of the photoperiod. Third, the cellular basis for growth induced by extending the photoperiod was different from that observed with GA₃. It was concluded that light and gibberellins are both important in the overall regulation of petiole growth, but act through independent mechanisms.     

Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA_{1,9,12,15,19,20,24,29,44,51,53}. Traces of GA₈ and GA₂₅ were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA₁₉ and GA₄₄ were most abundant, while GA_{12,15,20,24,29,53} occurred in lesser amounts. Only small amounts of GA_1,9,51 were present. The levels of GA₈ and GA₂₅ were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA_{1,8,19,20,29,44,53}) and another whose members are either nonhydroxylated (GA_{9,12,15,24,25}) or lack a C-13 hydroxyl group (GA₅₁). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA₁₂: an early C-13 hydroxylation pathway, leading ultimately to GA₁ and GA₈ and a C-13 deoxy pathway culminating in the formation of GA₉ and GA₅₁.     

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR₁) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA₃, was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA₃. Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.     

Determination of the Cellular Mechanisms Regulating Thermo-Induced Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermo-induced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A₃ completely mimicked the effects of a 4-week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain.     

Role of Gibberellins in the Environmental Control of Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual that requires a cold treatment for the induction of stem elongation. An inbred line was selected in which no stem elongation was observed in plants grown for 6 months at 21°C regardless of the prevailing photoperiod. Increased exposure time of plants grown initially at 21°C to cold (2°C) induced a greater rate of stem elongation when the plants were returned to 21°C. Moreover, longer cold treatments resulted in a greater maximum stem height and reduced the lag period for the onset of measurable internode elongation. The optimal temperature range for thermoinduced stem growth was broad: rates of stem growth in plants maintained for 4 weeks at either 2° or 10°C were virtually identical. However, a 4-week thermoinductive treatment at 15°C resulted in a greater lag period for the initiation of stem elongation and a decreased growth rate. The rate of cold-induced stem elongation was greater in plants subjected to long days than for plants subjected to short days following the cold treatment. Thus, photoperiod does not control the induction of stem elongation, but does regulate stem elongation in progress. Exogenous gibberellin A₃ (GA₃) was able to substitute for the cold requirement, but elicited a greater response in plants maintained under long days than short days. This indicates that photoperiod influences the plant's sensitivity to GAs. The GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride, inhibited low temperature-induced stem elongation, and this inhibition was completely reversed by exogenous GA₃. These results suggest that cold-induced stem elongation in field pennycress is mediated by some change in the endogenous GA status.     

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [²H]-ent-kaurenoic acid (KA) and [¹⁴C]-gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [²H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [²H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A₉ (GA₉) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA₉. In contrast, thermo- and noninduced leaves metabolized exogenous [²H]-KA into GA₂₀ equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [¹⁴C]-GA₁₂-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA₁₂-aldehyde.     

Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A₁, (GA₁), GA₉, and GA₂₀ caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA₅₃, or GA₈. Moreover, of the biologically active compounds, GA₉ was the most active followed closely by GA₁. In thermoinduced plants (4-week cold treatment at 6°C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA₉ was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA₂₀. No such increase in activity was observed for either GA_[unk] or GA₅₃. The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA₉ is the endogenous mediator of thermoinduced stem growth in field pennycress.     

Plasticity of reproductive components at different stages of development in the annual plant Thlaspi arvense L.

Summary This study examines the effect of different densities and the removal of all neighbours at different stages of development on all components of reproduction in the inbreeding annual Thlaspi arvense L. A 64-fold increase in density significantly reduced all repooductive components. The number of flower buds per plant was decreased most strongly; the order of decreasing plasticity among the other components was number of capsules per flower, individual seed weight, ovule number per capsule, flowers per flower bud and seeds per ovule. Removing neighbours at all stages of development increased seed yield of plants in comparison to the control without density reduction, but patterns of plasticity depended strongly on time of treatment. The main effect of the removal of neighbours at the vegetative stage was to increase the number of flowers per plant, but the number of ovules per capsule and seed weight increased also, and abortion of capsules decreased. Removing neighbours at the onset of flowering initially failed to affect flower number per plant, instead it resulted in a strong reduction of capsule abortion and an increase in seed weight. However, several weeks after flowering had initially ceased, fresh lateral inflorescences were produced, resulting in a second flush of reproduction. Removing neighbours at the stage of fruit ripening resulted at first only in an increase in seed witht, but later a second reproductive phase occurred. Fresh lateral branches were produced, but the apical meristem was also reactivated. The overall pattern of plasticity among all reproductive components in response to a removal of neighbours was the same as in response to density. The position of a capsule along the inflorescence influenced its number of ovules, the rate of seed abortion and the mean weight of seeds, with the type of effect depending on the developmental stage at which neighbours were removed. Significant negative correlations were found between the mean weight of seeds and the number of seeds in a capsule under all treatments.     

Distinctive effects of cadmium on glucosinolate profiles in Cd hyperaccumulator Thlaspi praecox and non-hyperaccumulator Thlaspi arvense

We investigated the hypothesis that continuous water application allows favorable and steady water content and hydraulic conductivity in the root zone, thus enabling higher water potential in the soil–root interface (ψ_root). Elevated ψ_root increases transpiration (T) and prevents yield loss due to stomatal closure or to low root osmotic potential that develops in response to low ψ_root. We assume further, that the advantage of continuous water application is more pronounced for young plants, where water uptake per root length and competition on resources in the root system is higher. We investigated this hypothesis by examining the average water content of the root zone and T as a function of time for sunflowers grown under varied irrigation frequencies experimentally and in a modeled simulations, and by solving for the necessary effective root length and ψ_root for each case. High frequency water application was shown to positively affect root water uptake efficiency and yield, especially when plants were young. Irrigation frequency affected growth through the water content in the bulk soil (θ_soil) which in turn affects ψ_root. A low θ_soil and coupled low hydraulic conductivity decreased T and yield. Moreover, a decreased θ_soil caused low ψ_root, inefficient allocation of energy and carbohydrates and eventual yield loss. It was likely that these phenomena were more pronounced with young plants due to higher water uptake per root length.     

Cadmium exclusion a key factor in differential Cd-resistance in Thlaspi arvense ecotypes

Differences in Cd accumulation and Cd tolerance between Thlaspi arvense ecotype Aigues Vives (AV) from a commercial grower in South France and ecotype Jena collected in the polluted urban area of Jena (Germany) were reported here. Ecotype Jena exhibited considerable Cd-tolerance. Shoot and root masses were unaffected and root elongation was even enhanced by exposure to 50 μM Cd. In contrast, growth of ecotype AV was severely affected by this Cd treatment. Ecotype Jena was much more efficient in excluding Cd from both roots and shoots than ecotype AV. Despite the efficient restriction of Cd transport from roots to shoots in Jena, this ecotype maintained high root to shoot transport of Zn and Fe under Cd exposure. Cd supply strongly decreased the activities of antioxidant enzymes in AV, while in the Cd resistant Jena these activities either remained unaffected (SOD, APX) or were increased (CAT) by Cd supply. In conclusion, naturally selected Cd-tolerance in Thlaspi arvense is due to efficient Cd exclusion. The mechanisms underlying exclusion of Cd from the shoots seem Cd-specific yet they did not affect the homeostasis of Fe and Zn in the shoots.     

Herbicide Chlorsulfuron Decreases Assimilate Transport Out of Treated Leaves of Field Pennycress (Thlaspi arvense L.) Seedlings

Treatment of field pennycress (Thlaspi arvense L.) leaves with the herbicide chlorsulfuron resulted in a decrease in the export of assimilate. Twelve hours after a spot application of 1 microgram, assimilate translocation was 70% of that in control leaves. In excised leaves treated with chlorsulfuron the total amounts of sugars and free amino acids were 150 and 170%, respectively, of the amounts in control leaves, 30 hours after herbicide treatment. The amount of sucrose was 247% of that in control leaves. The increase in the concentration of sucrose in the chlorsulfuron-treated leaves, combined with the absence of an effect of chlorsulfuron on carbon dioxide fixation, suggests that the decrease in assimilate transport is not due to an effect on the synthesis of assimilates, but rather to an effect on their movement out of the leaves. Supplying branched-chain amino acids to the field pennycress seedlings prior to the application of chlorsulfuron prevented the occurrence of the effects described.     

A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense

Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate–myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.     

Cadmium tolerance in Thlaspi caerulescens: II. Localization of cadmium in Thlaspi caerulescens

Tissue and cellular compartmentation of Cd in roots and leaves of the hyperaccumulator Thlaspi caerulescens was investigated, using energy-dispersive X-ray microanalysis. In roots, Cd was determined in cortex parenchyma cells, endodermis, parenchyma cells of the central cylinder and xylem vessels. In leaves, it was found in cells lying on the way of water migration from the vascular cylinder to epidermal cells, which is in line with passive Cd transport by the transpiration stream. The mechanisms of Cd-detoxification in roots seem to be localized both in apoplast (e.g. binding to cell wall compounds) and inside cells since Cd was accumulated in these two compartments. On the other hand, in leaves Cd was found only in electron-dense deposits inside vacuoles, which suggests that vacuoles are the main compartment of its storage and detoxification in these organs.     

增补UV-B辐射对菥蓂生理特性及次生代谢产物的影响

为了解药用植物菥蓂(Thlaspi arvense)在紫外线(UV-B)辐射增强下生长及响应规律,以2月龄菥蓂幼苗为研究对象,在自然光照基础上人工增补3.26μW·cm^-2(T1)和9.78μW·cm^-2(T2)2种不同辐射强度处理,以自然光照下生长的菥蓂幼苗为对照,研究菥蓂幼苗中光合指标和叶绿素荧光参数、光合色素(叶绿素a和b)含量、渗透调节物质(丙二醛、可溶性蛋白、可溶性糖和脯氨酸)含量、抗氧化酶(超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过氧化氢酶)活性和总黄酮、总酚和黑芥子苷等次生代谢产物含量对不同UV-B辐射强度的响应。结果表明：2种辐射强度下净光合速率、气孔导度、蒸腾速率、胞间二氧化碳摩尔分数、最大荧光(F_m)、PSⅡ最大光化学效率(F_v/F_m)和PSⅡ潜在光化学效率(F_v/F_o)均随辐射强度增大而降低;叶绿素a和叶绿素b、可溶性蛋白、可溶性糖和脯氨酸等渗透调节物质、超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过...     

Effects of an introduced mustard,Thlaspi arvense,on soil fungal communities in subalpine meadows

The subalpine meadows of the Rocky Mountains, USA, are at the advancing front of global change; however, little is known about the sensitivities of high-elevation soil fungal communities to ongoing ecological changes. Soil fungi are sensitive to abiotic and biotic environmental stressors, including climate change, soil disturbance, and the presence of introduced, non-native plants. Invasive plants in the Brassicaceae (mustard family) are known to alter fungal community structure, suppress arbuscular mycorrhizal fungi, and change their relationship with native plant hosts in forest ecosystems, but these phenomena have not been studied in the subalpine zone where non-native mustard plants are becoming established. Here, we investigated whether the presence of the introduced mustard plant, Thlaspi arvense, is associated with distinct properties of the whole fungal and arbuscular mycorrhizal fungal communities in subalpine meadow ecosystems. We observed clear differences in the composition, relative abundance of core taxa, and mean taxon relatedness of soil fungal communities in plots with T. arvense relative to those with only native vegetation. A suite of novel fungi were associated with T. arvense, and overall patterns of AMF phylogenetic diversity were drastically reduced in association with its presence. Our results suggest that T. arvense introduction impacts the soil fungal community, with potential implications for native plant communities and soil nutrient cycling in high elevation meadows of the Rocky Mountains.     

Nickel Localization in Seeds of the Metal Hyperaccumulator Thlaspi pindicum Hausskn.

Scanning electron microscopy and x-ray microanalysis were usedto study the localization of nickel in seeds of the hyperaccumulatingspecies Thlaspi pindicum Hausskn. (Brassicaceae). The metalwas preferentially accumulated in the micropylar area oppositethe radicle and in the epidermis of cotyledons. In seed sections,nickel was detected only in the embryo epidermis. Possible reasonsfor this characteristic distribution are discussed. Copyright2001 Annals of Botany Company Embryo, epidermis, hyperaccumulator, x-ray microanalysis, micropylar area, nickel localization, seeds, Thlaspi pindicum Hausskn     

Accumulation of Nickel by Excluder Thlaspi arvense and Hyperaccumulator Noccaea caerulescens upon Short-Term and Long-Term Exposure

Russian Journal of Plant Physiology - The ability to accumulate nickel (Ni) was compared in hyperaccumulator Noccaea сaerulescens F.K. Mey and excluder Thlaspi arvense L. after a...