The Effect of Epibrassinolide on Arabidopsis Seedling Morphogenesis and Hormonal Balance under Green Light

We studied the effect of 24-epibrassinolide (EB) on the levels of endogenous hormones and photomorphogenesis of Arabidopsis thaliana (L.) Heynh wild-type (Ler) and mutant (hy4) seedlings. This mutant is deficient in the cryptochrome 1 (CRY1) synthesis. CRY1, which is a product of the HY4 gene, is a blue light photoreceptor in wild-type plants, but is sensitive to green light as well. In dark-grown seven-day-old mutant seedlings, the ABA/zeatin ratio differed from this ratio in wild-type seedlings. Thehy4 mutant exhibited a lower zeatin and higher free-ABA contents, which could retard its hypocotyl growth in darkness. EB retarded the growth of hypocotyls in etiolated hy4 seedlings and enlarged their cotyledons more efficiently than in wild-type seedlings. Green light (GL) did not affect the growth of hypocotyls but enlarged cotyledons of hy4 seedlings, which might be associated with some increase in the level of free IAA and a considerable decrease in free ABA and also with a decrease in the cytokinin level in seedlings. The hy4 cotyledon response to GL depended evidently on photoreceptors other than CRY1. GL enhanced the effects of EB on the morphogenesis of both Ler and hy4 seedlings, which was coupled with changes in the balance of endogenous IAA, ABA, and cytokinins. We may suppose that EB is involved in the control of photomorphogenesis by interaction with endogenous hormones, which are involved in the transduction of a light signal absorbed by the GL photoreceptors.     

Maize seedlings produced from dry seeds exposed to liquid nitrogen display altered levels of shikimate pathway compounds

In light of climate change and risks of food insecurity, it is becoming increasingly important to preserve plant germplasm in genebanks. Storage of seeds, particularly via cryopreservation, is one of the most proficient methods for ex situ plant germplasm conservation. Whilst seed cryo-banking can have little, to no, or even beneficial effects on subsequent seedling vigor in some species, it can lead to a number of plant abnormalities (morphological and physiological). This study investigated the effects of maize seed cryopreservation on seedling growth (until 14 d) and levels of selected amino acids produced in the shikimate pathway, a major link between primary and secondary metabolism. Seed cryopreservation reduced FW in recovered seedlings, reduced caffeic acid (2.5-fold decrease), and increased levels of all other shikimate pathway–related compounds assessed: phenylalanine (2.9-fold increase), tyrosine (2.6-fold increase), and shikimic (2.1-fold increase) and protocathecuic (3.1-fold increase) acids in cotyledons. Our results suggest that maize seed cryopreservation results in seedlings that exhibit signs of an ‘overly’ efficient and caffeic acid–deficient shikimate pathway, possibly related to their reduced growth during a highly vulnerable growth stage. However, these metabolic abnormalities manifested most severely in the maternal (cotyledonary), as opposed to vegetative (roots, stems, and leaves), tissues and hence are likely to disappear when the seedlings shed the cotyledons and become completely autotrophic.

     

Evidence against the involvement of DNA synthesis in phytochrome-mediated photomorphogenesis

In order to clarify the relationship between photomorphogenesis and DNA replication we investigated the effect of continuous far-red or white light on the synthesis of DNA in the cotyledons and the hypocotyl of mustard seedlings between 36 and 108 h after sowing. The total DNA content of the cotyledons (about 2.2 pg cell^-1) did not significantly change during this period although long-term labeling experiments revealed newly synthesized DNA of nuclear, plastid, and mitochondrial origin. Light had no detectable effect on total DNA content and on the labeling of either DNA fraction. Histoautoradiography indicated that nuclear DNA synthesis was exclusively localized in dividing stomatal cells and in sieve tube companion cells undergoing endopolyploidization. The DNA content of the hypocotyl increased continuously but likewise showed no detectable effect of light. It is concluded that cell growth and differentiation during photomorphogenesis is independent of DNA synthesis.Abbreviation DABA 3,5-diaminobenzoic acid     

Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings

The role of abscisic acid (ABA) during early development was investigated in tomato seedlings. The endogenous content of ABA in particular organs was analyzed in seedlings grown in the dark and under blue light. Our results showed that in dark-grown seedlings, the ABA accumulation was maximal in the cotyledons and elongation zone of hypocotyl, whereas under blue-light, the ABA content was distinctly reduced. Our data are consistent with the conclusion that ABA promotes the growth of etiolated seedlings and the results suggest that ABA plays an inhibitory role in de-etiolation and photomorphogenesis in tomato.     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 μg N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 μg of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.     

Genetic interactions between the chlorate-resistant mutant cr 8 8 and the photomorphogenic mutants cop1 and hy5

The chlorate-resistant mutant cr88 is defective in photomorphogenesis, as shown by the phenotypes of long hypocotyls in red light and yellow cotyledons under all light conditions. A subset of light-regulated genes is expressed at subnormal levels in cr88. To analyze further the role that CR88 plays in photomorphogenesis, we investigated the genetic interactions between cr88 and mutants of two other loci affecting photomorphogenesis, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and LONG HYPOCOTYL5 (HY5). COP1 represses the expression of light-regulated genes in the dark, and HY5 inhibits hypocotyl elongation in the light. Using morphological, cellular, and gene expression criteria for epistasis analyses to position CR88 in the genetic hierarchy of the photomorphogenesis pathway, we determined that CR88 acts downstream of COP1 but in a branch separate from HY5. In the course of our analysis, we discovered that light causes extensive destruction of plastids in dark-grown cop1 seedlings and that cr88 prevents this destruction.     

The effects of DELLAs on growth change with developmental stage and brassinosteroid levels

There are two stages in photomorphogenesis. First, seedlings detect light and open their cotyledons. Second, seedlings optimize their light environment by controlled elongation of the seedling stem or hypocotyl. In this study, we used time‐lapse imaging to investigate the relationship between the brassinosteroid (BR) and gibberellin (GA) hormones across both stages of photomorphogenesis. During the transition between one stage and the other, growth promotion by BRs and GAs switched from an additive to a synergistic relationship. Molecular genetic analysis revealed unexpected roles for known participants in the GA pathway during this period. Members of the DELLA family could either repress or enhance BR growth responses, depending on developmental stage. At the transition point for seedling growth dynamics, the BR and GA pathways had opposite effects on DELLA protein levels. In contrast to GA‐induced DELLA degradation, BR treatments increased the levels of REPRESSOR of ga1‐3 (RGA) and mimicked the molecular effects of stabilizing DELLAs. In addition, DELLAs showed complex regulation of genes involved in BR biosynthesis, implicating them in BR homeostasis. Growth promotion by GA alone depended on the PHYTOCHROME INTERACTING FACTOR (PIF) family of master growth regulators. The effects of BR, including the synergistic effects with GA, were largely independent of PIFs. These results point to a multi‐level, dynamic relationship between the BR and GA pathways.     

Effects of methyl jasmonate on accumulation of flavonoids in seedlings of common buckwheat (Fagopyrum esculentum Moench)

The jasmonates, which include jasmonic acid and its methyl ester (MJ), play a central role in regulating the biosynthesis of many secondary metabolites, including flavonoids, and also are signaling molecules in environmental stresses. Synthesis of anthocyanins pigments is a final part of flavonoids pathway route. Accumulation of the pigments in young seedlings is stimulated by various environmental stresses, such as high-intensity light, wounding, pathogen attack, drought, sugar and nutrient deficiency. The anthocyanins take part in defense system against excess of light and UV-B light, and therefore it is probably main reason why young plant tissues accumulate enlarged levels of the pigments. The effects of exogenously applied MJ on level of anthocyanins, glycosides of apigenin, luteolin, quercetin and proanthocyanidins in seedlings of common buckwheat (Fagopyrum esculentum Moench) were studied. MJ decreased contents of all the found cyanidin glycosides and its aglycone in hypocotyls of buckwheat seedlings. However contents of particular anthocyanins in cotyledons of buckwheat seedlings treated with the plant hormone were not significantly different from the control. Applied doses of MJ did not affect levels of quercetin, apigenin and luteolin glycosides in the analyzed parts of buckwheat seedlings: cotyledons and hypocotyls. On the other hand, treatment of buckwheat seedlings with MJ clearly stimulated of proanthocyanidins biosynthesis in hypocotyls. We suggest that methyl jasmonate induces in hypocotyls of buckwheat seedlings the leucocyanidin reductase or anthocyanidin reductase, possible enzymes in proanthocyanidins synthesis, and/or inhibits anthocyanidin synthase, which transforms leucocyanidin into cyanidin. According to our knowledge this is the first report regarding the effect of methyl jasmonate on enhancing the accumulation of proanthocyanidins in cultivated plants.     

Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

In this study using biochemical approaches we identified two calcium-dependent protein kinases (CDPKs) named PnCDPK52 and PnCDPK56 in soluble protein extracts from seedlings of Pharbitis nil. Both enzymes phosphorylated the specific substrate histone III-S in the presence of Ca²⁺ and cross-reacted with antibodies against the CDPK. PnCDPKs exhibited quite different activity and protein levels during germination and successive stages of seedling growth. PnCDPK52 protein level was high in seeds and during germination, whereas PnCDPK56 increased in the next stages of seedling growth, being the dominant enzymes in mature seedlings, of the light- and dark-grown plant. In all cases both activity and accumulation of protein PnCDPK56 was higher in dark grown plants whereas exposure to light reduced both factors. When etiolated cotyledons were exposed to light, the activity of PnCDPK56 was reduced to the basal level within 5 h. Conversely, increasing activity of PnCDPK56 in cotyledons of green plants shifted to darkness was extremely rapid, reaching the maximum level after just 1 h of darkness and then gradually decreased. Further lengthening of the darkness to 16 h resulted in a strong increase in activity at 12 h. These data indicate that at least two isoforms of CDPK are involved in germination and seedling growth of P. nil. The differences in PnCDPKs strongly argue for the pleiotropic role of these isoforms. It seems that PnCDPK52 is associated with the germination process and PnCDPK56 with seedling growth. Moreover it suggests that activity of PnCDPK56 is controlled by light via the photoreceptor-dependent pathway.     

Metabolomics reveals organ-specific metabolic rearrangements during early tomato seedling development

Tomato seedlings (Solanum lycopersicum cv. MoneyMaker), grown under strictly controlled conditions, have been used to study alterations occurring in secondary metabolite biosynthetic pathways following developmental and environmental perturbations. Robustness and reproducibility of the system were confirmed using detailed statistical analyses of the metabolome. LCMS profiling was applied using whole germinated seeds as well as cotyledons, hypocotyls and roots from 3 to 9 days old seedlings to generate relative levels of 433 metabolites, of which 62 were annotated. Initial focus was given to the polyphenol pathway and several additional mass signals have been putatively annotated using high mass resolution fragmentation. Clear organ and developmental stage—specific differences were observed. Seeds accumulated saponin-like compounds; roots accumulated mainly alkaloids; cotyledons contained mainly glycosylated flavonols and; hypocotyls contained mainly anthocyanins. For each organ, the developmental changes in metabolite profiles were described by using linear mixed models. Across three independent experiments, 85 % of the metabolites showed similar developmental trends. This tomato seedling system has given us valuable additional insights into the complexity of seedling secondary metabolism. How metabolic profiles reflect an interplay between depletion of stored molecules and de novo synthesis and how the overall picture for this important crop plant contrasts to e.g. Arabidopsis are emphasised.     

Photomorphogenesis of the root system in developing sunflower seedlings: a role for sucrose

• The domestic sunflower (Helianthus annuus L. cv. ‘Giganteus’) has been used since the 19th century as a model plant for the study of seedling development in darkness and white light (WL) (scoto‐ versus photomorphogenesis). However, most pertinent studies have focused on the developmental patterns of the hypocotyl and cotyledons, whereas the root system has been largely ignored.
• In this study, we analysed entire sunflower seedlings (root and shoot) and quantified organ development in the above‐ and belowground parts of the organism under natural (non‐sterile) conditions.
• We document that seedlings, raised in moist vermiculite, are covered with methylobacteria, microbes that are known to promote root development in Arabidopsis. Quantitative data revealed that during photomorphogenesis in WL, the root system expands by 90%, whereas stem elongation is inhibited, and hook opening/cotyledon expansion occurs. Root morphogenesis may be mediated via imported sucrose provided by the green, photosynthetically active cotyledons. This hypothesis is supported by the documented effect of sucrose on the induction of lateral root initials in sunflower cuttings. Under these experimental conditions, phytohormones (auxin, cytokinin, brassinolide) exerted little effect on root and cotyledon expansion, and no hormone‐induced initiation of lateral roots was observed.
• It is concluded that sucrose not only acts as an energy source to fuel cell metabolism but is also a shoot‐derived signalling molecule that triggers root morphogenesis.

     

茉莉素介导的长春花生物碱次生代谢转录调控机制

萜类吲哚生物碱（terpeniod indole alkaloids, TIAs）是植物中产生的一类具有药理活性的次生代谢产物.药用植物长春花（Catharanthus roseus）因含有长春碱和长春新碱等重要的抗肿瘤萜类吲哚生物碱而成为研究TIAs次生代谢的主要模式植物.应用正、反向遗传学和各种代谢组学技术对长春花TIAs次生代谢途径及其调控进行了较深入的研究,相继鉴定了参与TIAs代谢途径调控的CrORCAs、CrMYCs、CrZCTs和CrWRKYs等转录因子,特别是发现茉莉素（jasmonates, JAs）介导TIAs生物合成的转录调控网络. 本文以长春花TIAs生物合成途径为模式,重点论述其代谢途径中的关键酶、参与调节的转录因子,尤其是茉莉素介导的调控网络及机制,解析植物中这些天然抗癌生物碱合成积累水平低的制约因素和组织细胞特异性,讨论基于这些新知识的长春花抗肿瘤TIAs代谢工程策略和工厂化绿色生产前景.     

Formation of isopentenyl diphosphate via mevalonate does not occur within etioplasts and etiochloroplasts of mustard (Sinapis alba L.) seedlings

Seedlings from the white mustard, Sinapis alba, grown under continuous far-red light exhibit enhanced plastid enzyme activities when compared with dark-grown seedlings (for review, see Mohr 1981). These activities are even more pronounced upon illumination with white light during the etioplast/chloroplast transformation. Etioplasts and etiochloroplasts from the cotyledons of such seedlings show high prenyl-lipid-synthesizing activities when [1-¹⁴C]isopentenyl diphosphate is used as the precursor. They lack, however, any enzymatic activities for the formation of isopentenyl diphosphate via the mevalonate pathway, i.e. hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase, which are present and easily detectable within the endoplasmic reticulum and cytoplasm. These results corroborate the view that the cytoplasm of the plant cell is the only site of isopentenyl-diphosphate formation via the mevalonate pathway.     

The specific responses to mechanical wound in leaves and roots of Catharanthus roseus seedlings by metabolomics

The mechanical wound is one of the unavoidable threats to survival of plants. More researchers focus on the effect of mechanical wound to the over-ground tissues. And the effects of wound to roots were frequently ignored, although it is an important organ for plant growth. In our studies, the metabolomics study was performed to reveal the mechanical wound effects in Catharanthus roseus on roots and leaves by combining gas chromatography-mass spectrometer (GC-MS), liquid chromatograph-mass spectrometer (LC-MS) and statistical analyses. The metabolic response of TIAs and PCs in plants to wound was most active at 0.5 h after treatment via Q value analysis. At this time point, then significantly responsive primary metabolites and specific secondary compounds (TIAs and PCs) were screened by PLS-DA score plot. In this case, the treatments of CK, LT (wound to leaves) and RT (wound to roots) were clearly distinguished. The targeted compounds include 8 sugars, 4 TIAs and 12 PCs and they displayed specific responses to CK, LT and RT treatments. Under RT group, plants invest more resources on the local responses using TIAs and the color reactions to regulate wound close using PCs. Whereas, LT group might lay emphasis on systemic responses via TIAs induced by SA (salicylic acid) and gallic acid. Our studies provide some basic data for further investigations of the defensive mechanism on roots treated by mechanical wound.     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 g N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 g of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.Scientific Contribution No. 1219 from the New Hampshire Agricultural Experiment Station.