Red coloration of apple (Malus x domestica) skin is an important determinant of consumer preference and marketability. Anthocyanins are responsible for this coloration, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. Regulation of expression of these genes is believed to be controlled by MYB transcription factors, and the MYB transcription factors involved in the activation of anthocyanin biosynthetic genes have been isolated in various plants. In the present study, we isolated and characterized a MYB transcription factor gene (MdMYBA) from apple skin. Characterization of MdMYBA demonstrated that (i) MdMYBA expression was specifically regulated depending on the tissue and cultivar/species; (ii) its expression level was much higher in a deep-red cultivar ('Jonathan') than in a pale-red cultivar ('Tsugaru'); (iii) when cauliflower mosaic virus 35S::MdMYBA was introduced into the cotyledons of apple seedlings by means of a transient assay, reddish-purple spots were induced, and MdMYBA also induced anthocyanin accumulation in reproductive tissues of transgenic tobacco; (iv) the expression of MdMYBA was induced by UV-B irradiation and low-temperature treatment, both of which are known to be important in the promotion of anthocyanin accumulation in apple skin; (v) MdMYBA bound specifically to an anthocyanidin synthase (MdANS) promoter region in a gel-shift assay; and (vi) MdMYBA was mapped to the near region of the BC226-STS (a1) marker for the red skin color locus (R(f)). These results suggest that MdMYBA is a key regulatory gene in anthocyanin biosynthesis in apple skin.

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To clarify the involvement of the arginine decarboxylase (ADC) pathway in the salt stress response, the polyamine titre, putrescine biosynthetic gene expression, and enzyme activities were investigated in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] in vitro callus under salt stress, during recovery after stress, and when ADC was inhibited by D-arginine, an inhibitor of ADC. Salt stress (200 mM NaCl) caused an increase in thiobarbituric acid-reactive substances (TBARS) and electrolyte leakage (EL) of the callus, which was accompanied by an increase in free putrescine content, during 7 d of treatment. Conjugated putrescine was also increased, but this increase was limited to the early stage of salt stress. Accumulation of putrescine was in accordance with induction of ADC activity and expression of the apple ADC gene (MdADC). When callus that had been treated with 200 mM NaCl was transferred to fresh medium with (successive stress) or without (recovery) NaCl, TBARS and EL were significantly reduced in the recovery treatment, indicating promotion of formation of new callus cells, compared with the successive stress treatment. Meanwhile, MdADC expression and ADC activity were also decreased in the callus undergoing recovery, whereas those of the callus under successive stress were increased. Ornithine decarboxylase (ODC) activity showed a pattern opposite to that of ADC in these conditions. D-Arginine treatment led to more serious growth impairment than no treatment under salt stress. In addition, accumulation of putrescine, induction of MdADC, and activation of ADC in D-arginine-treated callus were not comparable with those of the untreated callus. Exogenous addition of putrescine could alleviate salt stress in terms of fresh weight increase and EL. All of these findings indicated that the ADC pathway was tightly involved in the salt stress response. Accumulation of putrescine under salt stress, the possible physiological role of putrescine in alleviating stress damage, and involvement of MdADC and ADC in response to salt stress are discussed.

Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13-kDa protein, named RPP13-1, that was abundant in the pure phleom sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP13-1 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

Our studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

Partial cDNAs of myb-related regulatory genes were isolated from the tetraploid Kyoho grape ( Vitis labruscana: V. labrusca x V. vinifera) and the expression patterns of the corresponding genes were studied. Since MybA gene expression is closely related to coloring and/or ripening of the berry (expression increases strongly with the commencement of coloring and berry softening, and is detected only in berry skin and flesh), full-length cDNAs for the gene were isolated from a mature-berry cDNA library. Three different species of MybA were identified from the cDNA sequences. Delivery of these cDNAs to somatic embryos of grape led to the induction of reddish-purple spots and UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) gene expression in non-colored embryos. The UFGT transcript was not detected in control embryos, while other structural genes for anthocyanin biosynthesis were expressed in both control and pigmented embryos. In addition, introduction of the UFGT gene induced the same reddish-purple spots in embryos. In contrast, treatment with the leucoanthocyanidin dioxygenase (LDOX) gene failed to induce these spots. Our results strongly suggest that MybA genes are involved in the regulation of anthocyanin biosynthesis in the grape via expression of the UFGT gene.

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