Mechanism Underlying2,4-Dichlorophenoxyacetic Acid Maintaining Citrus Fruit Postharvest Quality

2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most effective antistaling agents for postharvest citrus fruits. However, considering the environmental and food security, its usage is still controversal. Illuminating the mechanism upon2,4-D preserving citrus fruit postharvest quality will give scientific clues for pursue new safer and effective preservatives. In this work, the changes in Olinda orange (Citrus sinensis (L.) Osbeck) fruit peel induced by2,4-D treatment were comprehensively analyzed by using gene microarray, protein2D-electrophoresis, high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), gas chromatography (GC), scanning electronic microscope (SEM) and GC-MS. To further obtain the early-responsive key genes regulated by2,4-D, the effects of2,4-D, IAA, GA3and24-epicastasterone on fruit quality and on gene expression in postharvest Satsuma Mandarin (Citrus Unshiu Marc. cv. Guoqing No.l) were determine and compare by using citrus genome microarrays. The main results were showed as follow:1. The macro-and micro-changes in Olinda orange fruit induced by2,4-D treatmentData of transcriptional profile showed that totally3,413genes were differentially expressed between control and2,4-D treated samples, and they were classified into34MapMan catalogues. Genes involve in the biological progresses of protein biosynthesis-folding-degradation, RNA transcription, transport, secondary metabolism, and hormone metabolism were significantly over-represented. Among these enriched categories, genes related to transcriptional factors (mainly AP2/ERF, WRKY and NAC family members), transport and hormone metabolism were over-represented and up-regulated at early stage (within24h) after treatment which suggested that these genes may mainly participated in exogenous2,4-D absorbing and transporting, then triggering the perception and signal transduction, then set up the down-stream cascade responses. Stress defense genes were up-regulated, while cell wall metabolism genes were down-regulated at late stage (after24h). However, the secondary metabolism, phenylpropanoids, lignin and epicuticular wax biosynthesis related genes in particular, over-represented over all time points. These genes might be the results of cascade responses of the earlier biological progresses and performed direct functions on fruit quality preservation.Data of comparative proteomic profile showed that abundance of99protein spots were significantly changed by2,4-D treatment,76of them were successfully identified and classified into11categories. Consistent with the transcriptional profile, the expressions of proteins implicated in stress responses, hormones metabolism and signal transduction, protein biosynthesis-folding-degradation, as well as sugar transformation and TCA were significantly altered at the post-transcriptional level, which were included in the4most abundance categories and take account25%,12%,12%and12%of the total differentially expressed proteins, respectively.Hormone levels detected by HPLC-MS/MS showed that ABA, SA and2,4-D residual significantly accumulated, while ethylene production (detected by GC) decreased after2,4-D treatment. Total wax coverage on fruit surface has no change after treatment, however, both ultrastructure and proportional composition of cuticle wax changed. The surface of control fruit was rough and accompanied with crystal waxes and cracks on it. Epicuticular wax was squamous and loosely covered on the cuticle. The margins of stomatal guard cells were obscured for they were heavily embedded within cuticular wax, but the stomatal apertures could be clearly observed. In contrast, the treated fruit surface was relatively smooth and epicuticular wax was tightly covered on the cuticle. Cracks disappeared and crystalline wax became smaller. The stomatal apertures swelled out and the pores were clogged by waxes. Aldehyde and alkane, the two major components of total cuticular wax, respectively increased and decreased in treated fruit waxes. In addition,500mg/L2,4-D treatment significantly reduced the decay incidences and respiration rate but had no effect on fruit weight loss at years of2010and2012. lignin and water content in fruit peel were also increased,In conclusion,2,4-D retard fruit senescence by remodeling endogenous hormone balance and activating their signal transduction, then improving the ability of stresses defense through up-regulating defense-related genes and proteins.2. Evaluation the effects of2,4-D, IAA, GA3and24-epicastasterone on postharvest Satsuma Mandarin (Citrus Unshiu Marc. cv. Guoqing No.1) fruit quality and the comparative transcription profiles analysis of early responsive genes induced by these4hormonesThe effects of200mg/L and500mg/L2,4-D,50mg/L IAA,50mg/L GA3, and5mg/L24-epiCS on the external and internal quality of postharvest satsuma mandarin were evaluated. Compared with control, fruit rot rate was remarkably inhibited by each hormone treatment, however, the efficiency between hormones was significantly different. Efficiencies of2,4-D and IAA (rot rate<4%) were better than that of GA3and24-epiCS (rot rate<14%). Fruit texture was increased by2,4-D within the first4weeks and by GA3at late3weeks during the6weeks storage period. IAA and24-epiCS significantly reduced peel texture, fruit weight loss and peel water content. These4hormones have relatively little impact on fruit internal quality. SSC and ascorbic acid contents showed non obvious change after treatment. Though it was reduced by IAA,200mg/L2,4-D, and GA3, TA in juice displayed no changes after24-epiCS and500mg/L2,4-D treatment.Data of comparative transcription profile showed that812,386,864and391probe sets were differentially regulated by2,4-D, IAA, GA3and24-epiCS at1h post treatment, respectively. The up-regulated genes were more than down-regulated genes, and they take account about63.4%to89.2%of total differential gene regulated by each hormone. Totally,1214probe sets were differential regulated by one or more hormones, among them177were co-regulated by all4hormones. The similarity between2,4-D and GA3, or between IAA and24-epiCS were higher than71%. The differentially expressed genes regulated by each hormone were classified into11functional groups based on MAP-MAN categories. Statistics by hypergeometric distribution algorithm showed that genes implicated in RNA transcription, signal transduction over-represented, and transcription factors of AP2/EREBP, WRKY, and C2C2(Zn) CO-like transcription factor family were enriched in all hormone treatments, indicated that these genes might mediate crosstalk among the4hormones signal pathways.All the5over-represent genes categories induced by IAA treatment were nonspecific, suggest that the IAA signal pathway could interact with signal pathway of2,4-D or GA3or24-epiCS by these similar functional genes. Large amount of genes related to protein fate were regulated by2,4-D and GA3. Secondary metabolism related genes were specially regulated by24-epiCS and GA3. Expression of cell wall metabolism related genes were significantly changed by2,4-D, GA3and24-epiCS, other than IAA. Lipid metabolism genes were specially regulated by2,4-D. photosynthesis related genes were specially regulated by24-epiCS. These specially expressed genes might implicated in the special function of its regulating hormone. Genes in ethylene signal pathway such as ERF2, ERF9, CBF2, CBF1, CBF4, DREB1D and RAV2were simultaneously regulated by the4hormones, indicated that ethylene signal play key roles in hormone crosstalk. The rapid and comprehensive regulation of multiple hormone signaling by2,4-D treatment within1h including IAA, ABA, ethylene, JA and24-epiCS, and the special regulation of SA, GA3as well as lipid metabolism genes might involve in the early signaling transduction process of2,4-D as an high efficient preservative of citrus fruit.