Induction of anthocyanin pigments in callus cultures of <Emphasis Type="Italic">Rosa hybrida</Emphasis> L. in response to sucrose and ammonical nitrogen levels

The influence of varied concentrations of sucrose and ammonical (NH₄⁺) nitrogen on in vitro induction and expression of anthocyanin pigments from Rosa hybrida cv. ‘Pusa Ajay’ was investigated. Of two explants (petal and leaf discs) selected and cultured under two different conditions (light and dark), leaf discs were found to be most suitable for callus initiation. Profuse and early callus induction was observed when leaf discs of rose were cultured under total dark conditions on solid Murashige and Skoog (MS) medium supplemented with 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Early pigment initiation, enhancement and maximum anthocyanin production from calluses were recorded when leaf discs were cultured on Euphorbia millii (EM) medium supplemented with 7% sucrose compared with calluses cultured at 4% sucrose concentration under 16/8 h (light/dark) photoperiod regime. Reducing the concentration of NH₄⁺ nitrogen in the solid MS medium led to slight improvement in anthocyanin production in rose leaf calluses.     

Identification and characterization of genes induced for anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots using suppression subtractive hybridization, cDNA microarrays, and RNAi techniques

Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.     

Effects of Exogenous Indole Butyric Acid and Callus Formation on the Anti-oxidant Activity, Total Phenolic, and Anthocyanin Constituents of Mulberry Cuttings

In order to evaluate the effects of exogenous indole butyric acid （IBA） and callus formation on the antioxidant activity, total phenolics, and anthocyanin constituents of Morus nigra L. and M. alba L. cuttings, we investigated the variations before and after the treatment. The results indicate that anti-oxidant ability, total phenolic, and anthocyanin constituents of the callus stems of both Morus species were higher than those of non-callus forming species. There were also increases observed in anti-oxidant ability, total phenolic, and anthocyanin constituents of calli treated with IBA （1 000-3 000 mg/L）.     

Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas

The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent.     

Effect of ABA upon anthocyanin synthesis in regenerated torenia shoots

To elucidate the mechanism of anthocyanin synthesis induction concomitant with chlorophyll degradation, we established a system in which anthocyanin synthesis and degradation of chlorophyll in regenerated torenia (Torenia fournieri) shoots was induced on medium containing 7% sucrose. Here, we studied the effect of several plant-growth regulators on anthocyanin synthesis and the degradation of chlorophyll in the torenia shoot regenerating system. Exogenous abscisic acid (ABA) could induce anthocyanin synthesis and chlorophyll senescence in regenerating torenia shoots on the medium containing a low concentration of sucrose (1.5%). We determined the changes in the amount of endogenous ABA in the regenerated shoots during the process of anthocyanin synthesis on the medium containing 7% sucrose. It was revealed that the 7% sucrose treatment elevated endogenous ABA levels before the induction of anthocyanin synthesis and chlorophyll degradation. However, while retransfer to the 1.5% sucrose medium resulted in a gradual decrease in the ABA level and a failure of induction of anthocyanin synthesis, normal shoot regeneration. These results suggest that changes in the amount of endogenous ABA may play an important role in the induction of anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots.     

Dihydroflavonol Reductase Activity in Relation to Differential Anthocyanin Accumulation in Juvenile and Mature Phase Hedera helix L

Juvenile phase English ivy (Hedera helix L.) plants accumulate anthocyanin pigment in the hypodermis of stems and petioles, whereas genetically identical plants of the mature phase do not. The objective of this work was to assess which enzyme(s) might limit anthocyanin accumulation in mature phase ivy. Leaf discs of both juvenile and mature phase ivy accumulated comparable levels of the flavonols kaempferol and quercetin, whereas only juvenile phase discs accumulated anthocyanin. The accumulation of quercetin, but lack of accumulation of leucocyanidin or anthocyanin in mature phase discs, suggested that mature discs lacked dihydroflavonol reductase activity. There was no detectable dihydroflavonol reductase activity in mature phase discs, whereas there was an induction of activity in juvenile phase discs in response to sucrose, or photosynthetically fixed carbon, and light as a photomorphogenic signal. Phenylalanine ammonia-lyase, an enzyme early in the anthocyanin biosynthetic pathway, was induced above its basal level by sucrose and light in discs of both phases of ivy, with greater activity in mature phase discs. Phenylpropanoids, a class of compounds that are precursors to flavonoids, accumulated in leaf discs of both phases, with greater levels in mature phase discs. These results indicate that the lack of dihydroflavonol reductase activity limits the accumulation of anthocyanin in mature phase tissue.     

Regulatory effect of stems on sucrose-induced chlorophyll degradation and anthocyanin synthesis in Egeria densa leaves

Detached green leaves of the aquatic plant Egeria densa showed chlorophyll degradation and turned red due to induced anthocyanin synthesis incubated in 0.1 M sucrose under continuous light for 7–10 days. If the leaves were placed in water, only chlorophyll degradation occurred and the detached leaves turned yellow. The levels of endogenous total carbohydrates increased in detached leaves cultured in the sucrose solution but only increased marginally in water. If the leaves were still attached to a piece of stem, with a node on either side of the single leaf whorl, then they did not accumulate anthocyanin in culture with 0.1 M sucrose. These leaves showed a similar increase in total carbohydrates and degradation of chlorophyll as detached leaves. Attached leaves, in which the midrib had been cut in situ, showed localized accumulation of anthocyanin in the leaf tissue distal to the cut in the midrib when cultured in 0.1 M sucrose. These results suggest that the stem plays a regulatory role in anthocyanin synthesis in attached leaves cultured in a sucrose solution but does not influence chlorophyll degradation or carbohydrate accumulation.     

外植体与不同理化因子对虎杖愈伤组织诱导及次生代谢产物形成的影响

考察了外植体、培养基及光照条件对中药植物虎杖愈伤组织形成及次生代谢产物生产的影响。总的来看,所有愈伤组织中总酚和总黄酮含量比原植物的含量高2—3倍;而蒽醌的含量比原植物中含量低。外植体对愈伤组织形成及次生代谢产物生产的影响很大,所考察的3种外植体中。叶外植的愈伤组织诱导率最高而源于根外植体的愈伤组织具有最好的次生代谢能力。所考察的6种培养基中,MS＋0．5mg／L 2,4-D＋1．0mg／L 6-BA和N6＋0．5mg／L 2,4-D＋1．0mg／L 6-BA无论对于愈伤组织的产生还是次生代谢产物的累积都有较优表现。光照对愈伤组织诱导及次生代谢产物产生有明显影响。但二者无规律性联系。     

Red leaf color as a warning signal against insect herbivory: Honest or mimetic?

The co-evolution theory for red leaf colors considers redness as a handicap signal against herbivory. We have examined whether the assumed signal is honest and, accordingly, costly, by seeking a correlation between anthocyanin and total phenolic levels in 11 plants exhibiting variation in the expression of the red character either between individuals or between modules on the same individual. Selection of total phenolics as a variable was based on their assumed anti-herbivore function and on their common biosynthetic origin with anthocyanins. Plants with young or senescing red leaves were tested. Confirming evidence was found in senescing leaves, where in three out of the four studied species a significant and strongly positive correlation between signal strength (redness) and actual defensive potential (total phenolics) was found, rendering the signal both honest and costly. In young, developing leaves a significant, yet weakly positive correlation was found only in three out the seven examined species. Accordingly, the handicap signal hypothesis may be questioned in the case of young leaves. Hence, young leaf redness fits more to the alternative hypotheses that red leaf color is less easily perceived by folivorous insect photoreceptors or that red leaf color undermines insect camouflage. These hypotheses do not demand an increased chemical defensive potential.     

The effect of phosphate,nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of solanum laciniatum

In leaf derived callus cultures of Solanum laciniatum Ait. both phenolics and solasodine concentrations increased when medium phosphate or nitrogen concs. were reduced to one-eighth or when sucrose concentration was increased from 3 to 4–8 %. Under these conditions growth was reduced and final FW:DW fell. Growth was inhibited by sucrose depletion and nitrogen supple -mentation. On additional nitrogen the concentrations of phenolics and protein significantly increased, FW:DW was reduced and solasodine concentration was unaffected. In seedling derived cultures phosphate depletion resulted in a significant increase in phenolics concentration, an inhibition of growth and a rise in solasodine concentration.     

Metabolic interconnectivity among alternative respiration,residual respiration,carbohydrates and phenolics in leaves of Salvinia minima exposed to Cr(VI)

Floating and submerged leaves of the aquatic fern Salvinia minima were used to analyze a metabolic interconnectivity among mitochondrial alternative respiration, residual respiration (R_resp), carbohydrate metabolism and soluble phenolics (SP) accumulation occurring under Cr(VI) stress. Treatment with Cr enhanced alternative pathway capacity (AP_cap) and (R_resp) in both leaf types. AP_cap/T_resp ratio revealed an increasing relative contribution of the alternative respiration to total respiration rate under Cr(VI) treatment. Sucrose content increased in Cr-treated leaves, but glucose and starch decreased. Enzyme profile showed that sucrose synthase (SS) rather than soluble acid invertase (AI) seems to be involved in sucrose metabolism of Cr-treated plants. Accumulation of SP showed a positive correlation with both AP_cap and R_resp in floating leaves. Decreases of SP in submerged leaves can be explained by an increased synthesis of polymerized phenolics. Results provide important new insights about influence of alternative and residual respirations on the synthesis of phenylpropanoid-derivative compounds. This work could also represent the first communication about involvement of the R_resp in defence mechanism of S. minima against Cr(VI) toxicity.     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Carbohydrate accumulation may be the proximate trigger of anthocyanin biosynthesis under autumn conditions in Begonia semperflorens

Many plant leaves appear red in the autumn, and many papers have focused on the environmental factors and role of anthocyanin in this process. However few papers have examined the substances that are induced during this process. We hypothesised that excess sugar accumulation directly induces anthocyanin accumulation under autumn conditions. Using two methods (restricting phloem movement and exogenous sucrose feeding), we found that both surplus photosynthate and exogenous sucrose could induce anthocyanin biosynthesis, corresponding to up‐regulation of several enzymes involved in anthocyanin biosynthesis (phenylalanine ammonia lyase, chalcone isomerase, dihydroflavonol 4‐reductase and flavonoid 3‐O‐glucosyl transferase) and in transport (glutathione S‐transferase). Our results suggest that excess carbohydrate may be the proximate trigger for induction of anthocyanin biosynthesis in autumn, but only when carbohydrates are accumulated for storage.     

Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins

We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.     

The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures.

The effect of different concentrations of growth regulators and sucrose on anthocyanin production in cell suspension cultures of Camptotheca acuminata Decaisne (Nyssaceae) was described for the first time and qualitatively and quantitatively evaluated. Anthocyanin production was significantly greater in the presence of kinetin, compared to benzyladenine, with the greatest concentration observed in the presence of 2 microM kinetin. No significant differences in anthocyanin production were observed when comparing 2,4-dichlorophenoxyacetic acid to alpha-naphthaleneacetic acid, except when using 2 microM, 2,4-dichlorophenoxyacetic acid, which resulted in greater anthocyanin production. High sucrose concentration enhanced the production of anthocyanins. Based on the absence of anthocyanin production in the dark, we concluded that light was essential for stimulating anthocyanin production. The optimised medium consisted of: 2 microM kinetin, 2 microM 2,4-dichlorophenoxyacetic acid and 292 mM sucrose. HPLC/DAD and HPLC/MS analyses revealed that the main anthocyanin was Cy 3-O-galactoside and that the minor derivative was Cy 3-O-glucoside.     

Effects of Explants,Medium Formulations and Light on Callus Induction and Secondary Metabolites Accumulated in the Calli of Polygonum cuspidatum (Polygonaceae)

Callus initiation of a Chinese traditional medical plant , Polygonum cuspidatum Sieb. et Zucc (Polygonaceae) was studied, with a focus on the factors that could influence the callus induction ability and secondary metabolites accumulation. In comparison with the parent explants, the levels of total phenolics and flavones were 2 to 3-fold higher and the levels of anthraquinones (AQ_s) were lower in all calli induced. Different explants exhibited great variability in callus induction rate and secondarymetabolite accumulation. Among the three explants tested, leaf explants were with the best capacity in callus induction, while root explants accumulated the highest levels in secondary metabolites. Two media of six tested, MS and N6 respectively plus 0.5 mg/L 2, 4-dichlorophenoxy acetic acid and 1.0 mg/L 6-benzylaminopurine, showed the best performances in both callus growth and secondary metabolite accumulation. Illumination shower a remarkable effects on both callus induction and secondary metabolite accumulation, but no regular pattern was observed.     

蔗糖添加对杉木低磷胁迫响应和蔗糖代谢的影响

为了揭示低磷胁迫下蔗糖对杉木低磷胁迫响应和蔗糖代谢的影响,选用两种不同磷效率杉木家系M32和M28进行低磷胁迫下的蔗糖添加试验,分析蔗糖添加对低磷胁迫下杉木形态特征、生理特性和低磷诱导相关基因表达的影响。结果表明：蔗糖添加促进了低磷胁迫下杉木苗高、根长、根表面积、根平均直径、根体积、根叶组织蔗糖含量和根叶组织无机磷含量的增加,但仍明显低于正常供磷处理下添加蔗糖处理的杉木增量。低磷促进杉木叶中花青素的积累,而正常供磷和低磷胁迫下的蔗糖添加处理都显著促进了叶片花青素含量的增加。随着胁迫时间的延长,M28与M32在根、叶组织的蔗糖含量存在显著差异,且M28根叶组织中的蔗糖合成酶活性和蔗糖磷酸合成酶活性都高于M32。蔗糖合成酶ClSuSy在M28和M32根系中受低磷胁迫诱导下调表达,但蔗糖添加处理明显诱导ClSuSy表达量升高,M28在正常供磷并添加蔗糖处理下的ClSuSy表达量显著高于其它处理。蔗糖转运蛋白SUT4、磷转运蛋白ClPht1;4、紫色酸性磷酸酶PAP1和PAP11在M28和M32根系中总体上受低磷胁迫诱导上调表达,且受蔗糖添加处理诱导下调表达。低磷胁迫下,添加或不添加蔗糖处理的M32根系SUT4的表达量均在15d时显著升高,并在45d时回落到正常水平。ClPht1;4和PAP1在低磷胁迫15d的表达量显著高于45d时的表达量,且ClPht1;4在M32根系中的表达量远高于M28。本研究表明,蔗糖对杉木低磷胁迫响应和糖代谢有重要的影响作用,低磷胁迫下添加蔗糖处理能够在一定程度上缓解杉木低磷胁迫响应。