Long-distance phloem transport of glucosinolates in Arabidopsis

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.     

Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus): II. Profiles and tissue-water concentrations in vegetative tissues and developing pods

The concentrations of glucosinolates in the tissue water of leaves, stems, floral buds and developing pods were measured at defined stages of development in four oilseed rape cultivars known to have different seed glucosinolate concentrations (Bienvenu, Ariana, Cobra and Capricorn). Five alkenyl, two aromatic and three indolyl compounds were identified in the vegetative and reproductive organs. Substantial differences developed in the profiles of compounds present during vegetative growth. The 2-hydroxy-3-butenyl glucosinolate was primarily associated with developing and mature seeds and germinated seedlings. The 4-pentyl glucosinolate occurred mainly during the later stages of vegetative growth in spring, when leaves contained higher concentrations than stems, and during the early stages of flowering. The 2-phenylethyl and 3-indolymethyl glucosinolates were present earlier in vegetative growth when similar concentrations were present in leaves and stems. Differences between cultivars in the tissue-water concentrations of individual glucosinolates were small during vegetative growth. The leaves of Cobra and Capricorn had smaller concentrations of the 3-pentyl glucosinolate than Bienvenu and Ariana especially early in spring, and the stems of Capricorn had smaller concentration of the 3-pentyl and 2-phenylethyl glucosinolates. Greater differences between cultivars developed during pod growth and involved large increases in the concentrations of the 2-hydroxy-3-butenyl and 3-butenyl glucosinolates, especially in Bienvenu and Ariana. The implications of these changes in the types and concentrations of glucosinolates in the different organs for the incidence of pests and diseases are discussed.     

Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus

The uptake of radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) by protoplasts isolated from leaves of Brassica napus was detected using silicone oil filtration technique. The uptake was pH-dependent with higher uptake rates at acidic pH. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of p-OHBG, which was inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone, indicating that the uptake is dependent on a proton motive force. Dissipation of the internal positive membrane potential generated a small influx as compared with that seen for pH gradient (DeltapH). Kinetic studies demonstrated the presence of two uptake systems, a saturable and a linear component. The saturable kinetics indicated carrier-mediated translocation with a K(m) of 1.0 mm and a V(max) of 28.7 nmol/microl/h. The linear component had very low substrate affinity. The carrier-mediated transport had a temperature coefficient (Q(10)) of 1.8 +/- 0.2 in the temperature range from 4-30 degrees C. The uptake was against a concentration gradient and was sensitive to protonophores, uncouplers, H(+)-ATPase inhibitors, and the sulfhydryl group modifier p-chloromercuriphenylsulfonic acid. The carrier-mediated uptake system had high specificity for glucosinolates because glucosinolate degradation products, amino acids, sugars, or glutathione conjugates did not compete for p-OHBG uptake. Glucosinolates with different side chains were equally good competitors of p-OHBG uptake, which indicates that the uptake system has low specificity for the glucosinolate side chains. Our data provide the first evidence of an active transport of glucosinolates by a proton-coupled symporter in the plasma membrane of rape leaves.     

Precociously germinating rapeseed embryos retain characteristics of embryogeny

We compared the germination of Brassica napus L. embryos at three stages of development-mid-cotyledon, maturation and mature dry-to determine at which stage they acquired the capacity for normal germination and seedling development. Embryos were removed from the seed and cultured on hormone-free medium, allowing them to germinate. The transition from embryogeny to germination was monitored both morphologically and biochemically, using synthesis of 12 S storage protein as a marker of embryogeny. The mature embryos (dry seeds) set the standard for normal seedling development: radicle emergence, hypocotyl extension and cotyledon expansion occurred within 2 d and true leaves were formed within a week of germination. Rocket immunoelectrophoresis indicated that the storage proteins in seedlings from mature dry embryos were completely degraded within a week. In contrast, the midcotyledon-stage embryos appeared to germinate abnormally, retaining many embryonic characteristics. Although the roots emerged, the hypocotyls did not elongate and secondary cotyledons instead of leaves were formed at the shoot apex. Also, the seedlings continued to synthesize and accumulate storage proteins. The maturation-stage embryos did develop into normal-looking seedlings, but complete degradation of storage proteins required several weeks, presumably reflecting continued synthesis and turnover. We conclude that embryogenic and germination-specific processes can occur concurrently and that the capacity to develop as normal seedlings is acquired gradually during the maturation process.Abbreviations dpa days post anthesis - EDTA ethylenedi-aminetetraacetic acid - FW fresh weight     

Glucosinolates and Glucosinolate Degradation in Seeds from Turnip Mosaic Virus-Infected Rapid Cycle Brassica campestris L. Plants

The effects of turnip mosaic virus infection on glucosinolatelevels and catabolism was studied in the mature seeds of rapidcycling Brassica campestris L. plants. Disease infection alteredthe total and soluble N concentrations in seeds. Infected plantssuffered growth reduction and increased concentration of severalseed glucosinolates compared to non-infected plants. The activityof partially purified seed myrosinase was not affected by maternalplant infection. Systemic viral infection did not induce qualitativeor quantitative alterations in the seed glucosinolate degradationprofile. Two major breakdown products, 3-butenylisothiocyanateand 1-cyano-3, 4-epithiobutane, were identified during autolysisand were, presumably, derived from 3-butenyl-GS, the most abundantseed glucosinolate. These results indicate that the glucosinolateconcentration and profile, but not glucosinolate catabolism,is affected in seeds produced on turnip mosaic virus-infectedrapid cycling plants. Key words: Glucosinolates, turnip mosaic virus, Cruciferae     

Cessation of assimilate uptake in maturing soybean seeds

In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [¹⁴C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [³H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [³H]glutamine to ethanolinsoluble components, but converted little exogenous [¹⁴C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability.     

Identification and characterization of a sucrose transporter isolated from the developing cotyledons of soybean

Reduced carbon produced in mature leaves is distributed throughout plants in the form of sucrose. Sucrose transporter proteins (SUT) play a crucial role in transporting sucrose. We isolated a cDNA encoding a sucrose transporter, GmSUT1, which is expressed in the developing cotyledons of soybean (Glycine max). [14C]sucrose uptake assays demonstrate that GmSUT1 has a K(m) of 5.6mM and a V(max) of 5.8 nmol sucrose min(-1)(mg cells)(-1), which are similar to those of the low-affinity-high-capacity sucrose transporter family. GmSUT1 protein accumulates gradually during cotyledon development, correlating with increasing sucrose levels in the maturing cotyledons. Collectively, these data suggest that GmSUT1 plays an active role in the movement of sucrose into the developing seeds.     

Maternal Effect on Embryogenesis in Tobacco Overexpressing Rice Phytochrome A*

When analyzing tobacco lines overexpressing various types of rice phytochrome A, we observed seedlings with fused cotyledons. Phytochrome A is a member of the phytochrome family of plant red/far-red absorbing photoreceptors. Reciprocal crossings with wild-type tobacco indicated that this abnormal phenotype was maternally inherited. Mother plants that were expected to produce abnormal seedlings, were raised under different conditions and seeds collected separately from individual capsules. The frequency of abnormal seedlings increased in seeds from later flowers but decreased when mother plants were raised under continuous white light. The interaction of the overexpressed phytochrome A proteins with endogenous plant hormones might be responsible for cotyledon fusion. The abnormal phenotype could partially be recovered by application of gibberellic acid to intact flowers at the time of pollination. In contrast, the synthetic auxin NAA slightly enhanced the degree of cotyledon fusion. Even wild-type tobacco seedlings exhibited partial fusion of cotyledons if flowers were treated with 2,4-D and occasionally the shoot apex was replaced by a second root. Application of GA₃ to the flowers, in contrast, impaired the development of the radicule. These observations are discussed with respect to maternal effects in plant embryogenesis.     

Damage to oilseed rape seedlings by the field slug Deroceras reticulatum in relation to glucosinolate concentration

Seedlings of nine commercial cultivars of oilseed rape were exposed to the field slug Deroceras reticulatum immediately after sowing in compost in trays. There was a small reduction in seedling numbers in the presence of slugs which was not related to glucosinolate concentration in seeds or seedlings. However, the number and leaf-area of seedlings with damage symptoms were strongly and inversely related to the total concentration of glucosinolates in seeds and one wk-old seedlings. The presence of barley seedlings as alternative food did not significantly affect this relationship. The glucosinolate concentration of seeds was closely correlated with that of wk-old seedlings. Analysis of individual glucosinolates in four cultivars spanning the range of concentrations found, showed that the concentration of most components declined as total glucosinolate concentration decreased. However, 2-phenyl ethyl-glucosinolate (gluconasturtiin) concentration tended to increase in seeds and 3-indolyl methyl-glucosinolate (glucobrassicin) increased in seedlings as total glucosinolate concentration decreased. Damage by slugs was inversely related to the concentration of those glucosinolates which decreased and was positively correlated with the two compounds which increased as total glucosinolate concentration decreased. The results support the hypothesis that glucosinolates in brassicas protect them from polyphagous herbivores, and, in particular, that an important function of glucosinolates in rape seeds is to protect seedlings from slugs. As glucosinolate concentrations of oilseed rape cultivars continue to decline, so the risk of slug damage to seedlings may well increase.     

The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns

We have analyzed expression conferred by two domains from the cauliflower mosaic virus (CaMV) 35S promoter and found different patterns in seeds, seedlings and seven week old plants. Expression from domain A (-90 to +8) is strongest in the radicle of the embryo, the radicle pole of the endosperm and in root tissue of seedlings and mature plants. Expression from domain B (-343 to -90) is strongest in the cells adjacent the cotyledon of the endosperm, in the cotyledons of the embryo and seedings and in the leaves and stem of mature plants. When both domain A and domain B are present expression is detectable in most tissues at all stages of development. Thus analysis of a constitutive promoter in transgenic plants can be used to identify cis elements that confer tissue specific and developmentally regulated expression.     

Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana

The glucosinolate content of various organs of the model plant Arabidopsis thaliana (L.) Heynh., Columbia (Col-0) ecotype, was analyzed at different stages during its life cycle. Significant differences were noted among organs in both glucosinolate concentration and composition. Dormant and germinating seeds had the highest concentration (2.5-3.3% by dry weight), followed by inflorescences, siliques (fruits), leaves and roots. While aliphatic glucosinolates predominated in most organs, indole glucosinolates made up nearly half of the total composition in roots and late-stage rosette leaves. Seeds had a very distinctive glucosinolate composition. They possessed much higher concentrations of several types of aliphatic glucosinolates than other organs, including methylthioalkyl and, hydroxyalkyl glucosinolates and compounds with benzoate esters than other organs. From a developmental perspective, older leaves had lower glucosinolate concentrations than younger leaves, but this was not due to decreasing concentrations in individual leaves with age (glucosinolate concentration was stable during leaf expansion). Rather, leaves initiated earlier in development simply had much lower rates of glucosinolate accumulation per dry weight gain throughout their lifetimes. During seed germination and leaf senescence, there were significant declines in glucosinolate concentration. The physiological and ecological significance of these findings is briefly discussed.     

Variation in the glucosinolate content of vegetative tissues of Chinese lines of Brassica napus L

The glucosinolate content of leaves, stems and roots of a range of Chinese oilseed rape (Brassica napus L.) breeding lines was analysed. Total content and spectrum of individual glucosinolates varied widely, and there was no correlation between seed and vegetative tissue glucosinolate content. Lines with low seed glucosinolates (00) did not necessarily have low glucosinolate content in vegetative tissues; nor did high seed glucosinolate lines always have high vegetative tissue content. There was no correlation between the glucosinolate content of leaf, stem, and root in any given line. It appears that glucosinolate synthesis and accumulation is under tissue-specific control, and the mutation which blocks accumulation of glucosinolates in seeds does not influence other tissues. The responses of these lines to elicitors was also examined. Methyl jasmonate and salicylic acid treatments produced increases in leaf indolyl and aromatic glucosinolates respectively. However, the extent of such increases differed widely between the lines, and there were other, less consistent, effects on other classes of glucosinolate. There seems to be greater variation in glucosinolate accumulation in rape than has previously been reported, and the lines described here have considerable potential for evaluating the effects of manipulating glucosinolate profiles on pest and disease interactions.     

Seed size, seedling morphology, and response to deep shade and damage in neotropical rain forest trees

To investigate the existence of coordinated sets of seedling traits adapted to contrasting establishment conditions, we examined evolutionary convergence in seedling traits for 299 French Guianan woody plant species and the stress response in a shadehouse of species representing seed size gradients within five major cotyledon morphology types. The French Guianan woody plant community has larger seeds than other tropical forest communities and the largest proportion of hypogeal cotyledon type (59.2%) reported for tropical forests. Yet the community includes many species with intermediate size seeds that produce seedlings with different cotyledonal morphologies. A split-plot factorial design with two light levels (0.8% and 16.1% PAR) and four damage treatments (control, seed damage, leaf damage, stem damage) was used in the shadehouse experiment. Although larger-seeded species had higher survival and slower growth, these patterns were better explained by cotyledon type than by seed mass. Even larger-seeded species with foliar cotyledons grew faster than species with reserve-type cotyledons, and survival after stem grazing was five times higher in seedlings with hypogeal cotyledons than with epigeal cotyledons. Thus, to predict seedling performance using seed size, seedling morphology must also be considered.     

Effect of salinity on seed germination,ion content and photosynthesis of cotyledons in halophytes or xerophyte growing in Central Asia

Aims We investigated the impact of salinity on seed germination, chlorophyll content, chloroplast structure and photosynthesis of the green embryos in desiccated seeds of the xerophyte Haloxylon persicum, xero-halophyte Haloxylon ammodendron and euhalophyte Suaeda physophora.Methods Seeds of H. persicum, H. ammodendron and S. physophora were collected from natural environment in Fukang, Xinjiang province. Pretreatment with 700 mM NaCl was carried out to stimulate the natural 'seed priming'; we analyzed the joint effect of salinity and different species on germination physiology and cotyledonal structure and photosynthetic function changes during germination and recovery stage.Important findings We found that seeds did not suffer ion toxicity for the two halophytes H. ammodendron and S. physophora, as evidenced by the high final germination after ungerminated seeds pretreated with 700 mM NaCl were transferred to distilled water, but the final germination of the xerophyte H. persicum was significantly lower than that of control. The Na + concentration in embryos increased under salinity for all species, while K + concentration decreased by salinity only for H. persicum and H. ammodendron, i.e. the concentration of K + in embryos of H. persicum and H. ammodendron decreased by 36% and 46%, respectively. For all species, whether dry intact seeds or cotyledons of dry seeds imbibed in deionized water and NaCl solution, had high chlorophyll content. Treatment with NaCl also caused chloroplast thylakoids to swell and chlorophyll content to decrease in seeds of H. persicum, but no significant change was observed in the more salt-tolerant species S. physophora and H. ammodendron. Fluorescence measurement showed that 700 mM NaCl decreased the Fv/Fm ratio of cotyledons in seeds for all species, especially for H. persicum and H. ammodendron. Photosynthetic oxygen releasing was detected from the seeds that were moistened with distilled water and 700 mM NaCl for 6 or 24 h and from the seeds that were initially moistened with 700 mM NaCl in darkness for 10 days, then transferred to distilled water for another 6 and 24 h. The results indicated that the chlorophyll in cotyledon of desiccated seed had photosynthetic function in early germination stage, even under high-saline condition. In addition, the photosynthesis of chlorophyll in the embryonic cotyledons of desiccated seeds during germination was similar to that in leaves of young seedlings for all species. In conclusion, the chloroplasts of the two halophytes were more salt resistant compared with the xerophyte H. persicum. The photosynthetic function of chlorophyll in cotyledons of mature seeds may be ecologically important for seedling development in early stage for plants growing in extremely saline or arid environments.     

Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors

Virus-induced gene silencing (VIGS) has great potential as a reverse-genetics tool in plant genomics. In this study, we examined the potential of VIGS in soybean seeds and the emergence stage of soybean plants using Apple latent spherical virus (ALSV) vectors. Inoculation of an ALSV vector (soyPDS-ALSV) carrying a fragment of the soybean phytoene desaturase (soyPDS) gene into soybean seedlings resulted in a highly uniform photo-bleached phenotype, typical of PDS inhibition, on the upper leaves throughout plant growth. The photo-bleached phenotype was also found on all immature pods, all seed coats, and about 50% embryos of seeds on soybean plants infected with soyPDS-ALSV. Infection with an ALSV vector (soyIFS2-ALSV) having a fragment of soybean isoflavone synthase 2 (soyIFS2) gene also led to a reduction of the levels of both soyIFS2- and soyIFS1- mRNAs and an isoflavone content in the cotyledons of about 36% mature seeds of infected soybean plants. Furthermore, VIGS of soyPDS was induced in the next generation plants by the seed transmission of soyPDS-ALSV. Thus ALSV vectors will be useful for studying gene functions in the reproductive stages and early growth stages, such as emergence and cotyledon stages, in addition to the vegetative stages of soybean plants.     

Ascorbic acid metabolism in ageing recalcitrant sal (Shorea robusta Gaertn. f.) seeds

Changes in ascorbate content and its enzymatic utilization pattern were studied in embryonic axes and cotyledons of sal seeds undergoing rapid loss of viability, at ambient conditions. Ascorbate levels were significantly higher initially in the embryonic axes (0.32 mg/g fresh weight) and cotyledons (0.21 mg/g fresh weight) of freshly mature, relatively hydrated (42.2% moisture content) and 100% viable sal seeds. It declined sharply as the tissues; embryonic axes and cotyledons, desiccated with absolutely no detectable amount in non-viable seeds (21% moisture content). Significantly strong correlation was obtained between desiccation of embryonic axes (r = 0.96) and cotyledon (r = 0.97) with loss of ascorbate levels and loss of germinability. Higher rates of ascorbic acid utilization (AAU) recorded in the embryonic axes of 100% viable seed declined sharply as the seed viability reduced due to desiccation below 36.8% moisture content. AAU was not detected in the cotyledons.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco).One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.