Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis,plant development,and carotenoid metabolism in fruits

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a‐overexpressing lines (OE‐CRY1a‐1 and OE‐CRY1a‐2). In comparison with wild‐type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE‐CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA‐seq, qRT‐PCR, and LC‐MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down‐regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE‐CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß‐carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.     

Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation

This paper reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue-light photoreceptor. Several point mutations were identified within the amino-terminal domain of CRY1—this region of CRY1 has high homology to photolyase and is likely to be involved in blue-light-mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy-terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue-light-mediated inhibition of hypocotyl elongation and we now report that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor—this is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated, and it is demonstrated that mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action.     

BBX16, a B‐box protein,positively regulates light‐induced anthocyanin accumulation by activating MYB10 in red pear

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

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.     

Arabidopsis ROOT HAIR DEFECTIVE3 is involved in nitrogen starvation-induced anthocyanin accumulation

Anthocyanin accumulation is a common phenom-enon seen in plants under environmental stress. In this study, we identified a new allele of ROOT HAIR DEFECTIVE3 (RHD3) showing an anthocyanin overaccumulat...     

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.Betacyanin biosynthesis in Phytolacca cells clearly shows a positive correlation with cell division, as the peak of betacyanin accumulation was observed at the log phase of batch cultures. Incorporation of radioactivity from labelled tyrosine into betacyanin also showed a peak at early log phase. Aphidicolin, an inhibitor of DNA synthesis, and propyzamide, an antimicrotubule drug, reduced betacyanin accumulation and inhibited the incorporation of radioactivity from labelled tyrosine into betacyanin at concentrations which were inhibitory to cell division. Both inhibitors reduced the incorporation of radioactivity from labelled tyrosine to 3,4-dihydroxyphenylalanine (DOPA), but the incorporation of labelled DOPA into betacyanin was not affected. These results suggest that the conversion of tyrosine to DOPA is coupled with cell division activity.In contrast, the anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Accumulation occurred at the stationary phase in batch cultures when cell division ceased. Aphidicolin or reduced phosphate concentration induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. Chalcone synthase (CHS) activity increased at the time of anthocyanin accumulation. Northern blotting analysis indicated that changes in CHS mRNA levels corresponded to similar changes in enzymatic activity. The pool size of endogenous phenylalanine was low during active cell division, but increased before anthocyanin began to accumulate and concomitantly with increasing levels of CHS mRNA. Exogenous supply of phenylalanine at the time of low endogenous levels induced the elevation of CHS mRNA and anthocyanin accumulation. These results indicate that the elevation of endogenous phenylalanine levels, when cell division ceases, may cause the increase in CHS mRNA levels, resulting in increased CHS activity and subsequently in anthocyanin accumulation in Vitis suspension cultures.Abbreviations CHS chalcone synthase - CHFI chalcone flavanone isomerase - DOPA 3,4-dihydroxyphenylalanine - PAL phenylalanine ammonia lyase     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants showed that CRY1-mediated blue-light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It was concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.