The color of fruit skin is an important quality parameter, and in many plants, it is the result of coordinative regulation of the anthocyanin pathway. To characterize the mechanism involved in fruit peel coloration of Yunnan red pear (Pyrus pyrifolia), we constructed a subtractive cDNA library using the suppression subtractive hybridization (SSH) technology. cDNA of red peel exposed to sunlight (for 2, 4, 6, and 8 days) was subtracted from that of white skin unexposed to sunlight. Over 100 differentially expressed ESTs were obtained, putatively involved in primary and secondary metabolism, stress, and defense response. Expression analysis using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) for 13 genes was performed with two pear cultivars, lightskinned 'Zaobaimi' and red-skinned 'Yunhong-1', which had been bagged and then exposed to sunlight for 0, 1, 2, 3, 5, and 7 days before harvest. This analysis showed that genes encoding for a metallothionein-like protein and a NADP-malic acid enzyme were constitutively expressed, whereas other selected genes were either down-or upregulated. Semiquantitative RT-PCR analysis for 7 anthocyanin biosynthetic pathway genes and 3 putative regulatory genes was also performed. Results showed that an R2R3 MYB transcription factor PyMYB10 was up-regulated in both the less-colored pear 'Zaobaimi' and well-colored red pear Yunhong-1 after the bag was removed, but that kinetics differed between cultivars. Other anthocyanin-related genes appeared to be coordinately regulated by the MYB-bHLH-WD40 complex. DFR and ANS genes seemed to be limiting factors for the peel coloration of less-colored pear 'Zaobaimi', while all biosynthetic steps are up-regulated by 7 days after bag removal in red fruit. This study suggests the regulation of red pear coloring is via differential effects of the MYB-bHLH-WD40 complex on the pear anthocyanin pathway genes.
Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in the present study. Our data indicated that an increase (120-130%) in MDH-specific activity in leaves led to an increase in malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared with the WT plants under the acidic (pH 4.5) conditions irrespective of 300 lM Al 3? stress absence or presence. After being exposed to 25 lM Al 3? in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance.
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