| Fengjie navel orange (Citrus sinensis Osbeck) is welcomed by consumers for its good taste and beautiful color. But it is prone to develop peel pitting, which has been described as a severe disorder with characteristics of the extensive collapsed areas of the flavedo (outer colored part of the peel) and part of the albedo (inner part of the peel) that becomes brown with time. It could affect the quality and decrease the market value of citrus fruits. Efforts have been made to elucidate the triggering factors of peel pitting, but little has been reported about the molecular mechanism. In our previous work, a suppression subtractive hybridization library of citrus peel pitting was established.Based on the sequences of cDNA fragments in the suppression subtractive library of citrus peel pitting, the full length cDNA of clone 7 and 27 were obtained from navel orange by Rapid Amplification of cDNA Ends. They were designated as CsCP and CsNAC, respectively. CsCP is 1409 bp in length, encoding a protein of 361 amino acids, with calculated molecular weight of 39.38 kDa and theoretical isoelectric point of 6.69.Sequence analysis showed that the CsCP protein had an N-terminal signal peptide predicted to translocate the preproprotein to the endoplastic reticulum and had high identity with other aleurain cysteine proteases. CsNAC is 1203 bp in length, encoding a protein of 305 amino acids, with calculated molecular weight of 35.2 kDa and theoretical isoelectric point of 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at N terminus and a highly variable region at C terminus, which are considered as the characteristic of NAC protein family. CsNAC protein was orthologous to Arabidopsis thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to stress stimuli.Quantitative RT-PCR determined the expression of CsCP was up-regulated during the development of postharvest peel pitting concomitant with senescence. The expression of CsNAC was also enhanced in the pitting peel compared to healthy peel. These results indicate that CsCP and CsNAC are peel pitting related genes and may play important roles in the development of citrus peel pitting.The expression patterns of CsCP and CsNAC response to abiotic stresses were examined by Northern blot analysis. The results showed that the expression of CsCP could be enhanced by low oxygen (3% O2) and high temperature (40oC), but repressed by no oxygen (0% O2), wounding and ethylene, which indicates that CsCP is involved in stress reponse. CsCP is the first cysteine protease whose mRNA expression has been found to be repressed by ethylene. The expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4oC) and exposure to ethylene also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40oC) but not affected by low oxygen (3%). It is indicated that CsNAC is not only a wound-inducible gene, but also an anoxia-related gene, which may play an important role in citrus postharvest peel pitting caused by abiotic stresses. CsNAC may be also involved in ethylene signaling.In this study, the full length cDNAs of two citrus peel pitting related genes were cloned, and their expression patterns during the development of peel pitting and response to abiotic stresses were also examined. Altogether, our results could provide an important theoretical basis for elucidating the molecular mechanism of citrus peel pitting.
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