Reactive oxygen species produced via plasma membrane NADPH oxidase regulate anthocyanin synthesis in apple peel

Main conclusion

Solar ultraviolet irradiation regulates anthocyanin synthesis in apple peel by modulating the production of reactive oxygen species via plasma membrane NADPH oxidase instead of other pathways. The synthesis of anthocyanin in apple peels is dependent upon solar irradiation. Using 3-mm commercial glass to attenuate solar UV-A and UV-B light, we confirmed that solar UV irradiation regulated anthocyanin synthesis in apple peels after exposing previously bagged fruit to sunlight. During sunlight exposure, UV attenuation did not affect the expression of MdHY5, MdCOP1, or MdCRY2, but significantly lowered plasma membrane NADPH oxidase activity and superoxide anion concentrations. UV attenuation also reduced the expression levels of MdMYB10, MdPAL, MdCHS, MdF3H, MdDFR, MdANS and MdUFGT1, UDP-glycose:flavonoid 3-O-glycosyltransferase (UFGT) activity, and local concentrations of anthocyanin and quercetin-3-glycoside. In contrast, exogenous application of hydrogen peroxide could enhance anthocyanin and quercetin-3-glycoside synthesis. Xanthophyll cycle pool size on a chlorophyll basis was higher but its de-epoxidation was lower under direct sunlight irradiation than that under UV-attenuating conditions. This suggests that reactive oxygen species (ROS) produced in chloroplast are not major contributors to anthocyanin synthesis regulation. Inhibition of plasma membrane NADPH oxidase activity lowered the production of ROS through this mechanism, significantly inhibited the synthesis of anthocyanin, and increased the total production of ROS in apple peel under direct sunlight irradiation, suggesting that ROS produced via plasma membrane NADPH oxidase regulates anthocyanin synthesis. In summary, solar UV irradiation regulated anthocyanin synthesis in apple peels by modulating the production of ROS via plasma membrane NADPH oxidase.     

Not all anthocyanins are born equal: distinct patterns induced by stress in Arabidopsis

Main Conclusion

Different abiotic stress conditions induce distinct sets of anthocyanins, indicating that anthocyanins have different biological functions, or that decoration patterns of each anthocyanin are used for unique purposes during stress. The induction of anthocyanin accumulation in vegetative tissues is often considered to be a response of plants to biotic or abiotic stress conditions. Arabidopsis thaliana (Arabidopsis) accumulates over 20 anthocyanins derived from the anthocyanidin cyanidin in an organ-specific manner during development, but the anthocyanin chemical diversity for their alleged stress protective functions remains unclear. We show here that, when grown in various abiotic stress conditions, Arabidopsis not only often accumulates significantly higher levels of total anthocyanins, but different stress conditions also favor the accumulation of different sets of anthocyanins. For example, the anthocyanin patterns of seedlings grown at pH 3.3 or in media lacking phosphate are very similar and characterized by relatively high levels of the anthocyanins A8 and A11. In contrast, anthocyanin inductive conditions (AIC) provided by high sucrose media are characterized by high accumulation of A9* and A5 relative to other stress conditions. The modifications present in each condition correlate reasonably well with the induction of the respective anthocyanin modification enzymes. Taken together, our results suggest that Arabidopsis anthocyanin profiles provide ‘fingerprints’ that reflect the stress status of the plants.     

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica)

Key message

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays.

Abstract

Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.     

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.     

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.

Abstract

Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

     

The role of anthocyanin in photoprotection and its relationship with the xanthophyll cycle and the antioxidant system in apple peel depends on the light conditions

The synthesis of anthocyanin, the xanthophyll cycle, the antioxidant system and the production of active oxygen species (AOS) were compared between red and non‐red apple cultivars, in response to either long‐term sunlight exposure (high light intensity) during fruit development, or to exposure of bagged fruits to lower light intensity late in fruit development. During fruit development of red and non‐red apples, the xanthophyll cycle pool size decreased much more in red apple peel late in development. With accumulation of AOS induced by long‐term sunlight exposure, enhancement of the antioxidant system was found. However, this change became significantly lower in red apple than non‐red apple as fruit developed, which might serve to accelerate the anthocyanin synthesis in red apple peel. When, late in fruit development, bagged fruits were exposed to sunlight, the accumulation of AOS was lower in red apple peel than in non‐red peel. This could be due to the higher anthocyanin concentration in the red peels. Meanwhile, compared with that in non‐red cultivar, the xanthophyll cycle and the antioxidant system in red apple peel were protected first but then down‐regulated by its higher anthocyanin concentration during sunlight exposure. In conclusions, red and non‐red apples peel possess different photoprotective mechanisms under high light conditions. The relationship between anthocyanin synthesis and the xanthophyll cycle, and the antioxidant system, depends on the light conditions that fruit undergoes.     

Photosynthetic costs and benefits of abaxial versus adaxial anthocyanins in Colocasia esculenta ‘Mojito’

Main conclusion

Anthocyanins in upper (adaxial) leaf tissues provide greater photoprotection than in lower (abaxial) tissues, but also predispose tissues to increased shade acclimation and, consequently, reduced photosynthetic capacity. Abaxial anthocyanins may be a compromise between these costs/benefits. Plants adapted to shaded understory environments often exhibit red/purple anthocyanin pigmentation in lower (abaxial) leaf surfaces, but rarely in upper (adaxial) surfaces. The functional significance of this color pattern in leaves is poorly understood. Here, we test the hypothesis that abaxial anthocyanins protect leaves of understory plants from photo-oxidative stress via light attenuation during periodic exposure to high incident sunlight in the forest understory, without interfering with sunlight capture and photosynthesis during shade conditions. We utilize a cultivar of Colocasia esculenta exhibiting adaxial and abaxial anthocyanin variegation within individual leaves to compare tissues with the following color patterns: green adaxial, green abaxial (GG), green adaxial, red abaxial (GR), red adaxial, green abaxial (RG), and red adaxial, red abaxial (RR). Consistent with a photoprotective function of anthocyanins, tissues exhibited symptoms of increasing photoinhibition in the order (from least to greatest): RR, RG, GR, GG. Anthocyanic tissues also showed symptoms of shade acclimation (higher total chl, lower chl a/b) in the same relative order. Inconsistent with our hypothesis, we did not observe any differences in photosynthetic CO₂ uptake under shade conditions between the tissue types. However, GG and GR had significantly (39 %) higher photosynthesis at saturating irradiance (A _sat) than RG and RR. Because tissue types did not differ in nitrogen content, these patterns likely reflect differences in resource allocation at the tissue level, with greater nitrogen allocated toward energy processing in GG and GR, and energy capture in RG and RR (consistent with relative sun/shade acclimation). We conclude that abaxial anthocyanins are likely advantageous in understory environments because they provide some photoprotection during high-light exposure, but without the cost of decreased A _sat associated with adaxial anthocyanin-induced shade syndrome.     

Flocculation phenomenon of Candida albicans by lysozyme

Young colonies of Sabouraud's glucose agar room temperature culture ofCandida species from human isolation were suspended in distilled water. The suspension was mixed with a solution of lysozyme and incubated in a 37° C water bath. Within 3–5 hours, various species ofCandida cells showed flocculation to varying degrees which occurred at varying periods of onset. Among sevenCandida species,Candida albicans andCandida stellatoidea showed the strongest flocculation, earliest onset and most solution clarity than did any other species.Candida stellatoidea was indistinguishable fromCandida albicans in its degree of flocculation, and in the clarity of solution.Candida species may be arranged in the following order according to their decreasing positivity in flocculation:

1. Candida albicans
2. Candida stellatoidea
3. Candida tropicalis
4. Candida krusei
5. Candida pseudotropicalis
6. Candida parapsilosis
7. Candida guilliermondii
8. Saccharomyces species may be placed afterCandida guilliermondii.

It seems possible to separate theCandida species into 3 groups by the rate of flocculation, and clarity of solution. Group I.Candida albicans andCandida stellatoidea. Group II.Candida tropicalis, C. krusei andCandida pseudotropicalis. Group III.Candida parapsilosis andCandida guilliermondii. Saccharomyces specimens (S. cerevisiae and others) were placed after group III.     

The effect of prey density on the functional and numerical reponses of three species of predatory mites

Functional and numerical responses of the predators:Phytoseiulus persimilis Athias-Henriot,Metaseiulus occidentalis (Nesbitt), andAmblyseius chilenensis (Dosse) [Acarina, Phytoseiidae] were observed at prey (Tetranychus urticae Koch [Acarina, Tetranychidae]) densities up to 300 prey/6.45 cm². Neither functional nor numerical response curves revealed any prey-predator interference effects, i.e.: the dome-shaped response curves (Holling, 1961), did not occur.