Exogenous ethylene treatment accelerated chlorophyll degradation in citrus fruits

The At2S3:RUBY results demonstrate that RUBY could be an effective marker for Arabidopsis transformation. Furthermore, betalain was not widely transported from the sites of synthesis to other tissues as we did not see any red color in leaves . We also expressed RUBY under the control of the Arabidopsis YUC4 promoter . YUC4, which encodes a key enzyme in auxin biosynthesis, was shown to express in small regions of embryos, leaves, and flowers. GUS signals were observed in leaf tips and apical region of a gynoecium in YUC4 promoter:GUS transgenic plants. We observed similar patterns of betalain production in YUC4:RUBY lines .The maturation of citrus fruit is coupled with significant changes both in peel color and in pulp sugar and acid content. The process is believed to be triggered and regulated by external and internal stimuli . Known plant hormones involved are ethylene, abscisic acid and gibberellins . Ethylene does not trigger an autocatalytic ethylene production and a corresponding respiration peak in the non-climacteric citrus fruits as it does in apple, raspberry container growing banana and other climacteric fruits. Yet, ethylene does trigger color changes in citrus fruit peel . Reduced ethylene production was found in some late-ripening mutants along with lower expression of ethylene biosynthesis genes, such as ACC synthase and ACC oxidase .

Ethylene involvement in citrus fruit maturation is also manifested by its regulatory effects on the expression of carotenoid biosynthetic genes, chlorophyllase genes and other fruit ripening– related genes . Increase in the ABA content was found to be concomitant with color development and carotenoid accumulation in peels of maturing citrus fruit . Upregulation of ABA biosynthesis and signaling genes were observed during citrus fruit maturation . Exogenous ABA treatment accelerated fruit coloration in Ponkan, whereas treatment with nordihydroguaiaretic acid, a synthetic ABA inhibitor, retarded fruit coloration and juice acid degradation . An ABA-deficient sweet orange [Citrus sinensis Osbeck] mutant, Pinalate, displayed slowed fruit degreening . The content of ABA and pigments was lower in the flavedo of a stay-green mutant of Ougan in comparison with that in the motherwood Ougan . Comparative transcriptional and proteomic analyses between late-ripening sweet orange mutants and their corresponding wild types also showed an essential role of ABA in citrus fruit maturation . In contrast to the promotive effects of ethylene and ABA, GA treatment delays the color break of citrus fruits. Applications of GA to the fruit of an early maturity pomelo-grapefruit hybrid could effectively retain the fruit color and prevent postharvest fruit senescence . Pre- or postharvest treatment of fruits with gibberellic acid retarded pigment changes in Satsuma mandarin . Auxins have been suggested to be important for the attainment of ability to ripen and for the coordination in the subsequent steps of the ripening process of both climacteric and non-climacteric fruits .

The content of indole-3-acetic acid , the primary natural auxin, was particularly high during fruit set and started to decline before the onset of fruit ripening . Application of exogenous auxins delayed the fruit ripening process in strawberry , tomato , banana and grape . However, an initial increase in IAA content is necessary for stimulating ethylene production and fruit maturation in climacteric fruits such as apple , plum , peach and pear . The IAA also regulates carotenoid biosynthesis by repressing the expression of phytoene synthase , ζ -carotene isomerase, phytoene desaturase and carotenoid isomerase, and promoting the expression of β-cyclase 1 and β-carotene hydroxylase during tomato fruit ripening . Impairment in IAA biosynthesis and signaling could also result in abnormal fruit ripening . A recent report showed that the exogenous application of NAA accelerated chlorophyll degradation and carotenoid accumulation in Satsuma mandarin fruit treated with two color-retarding agents, GA and prohydrojasmon . Extending fruit maturation will extend its marketable season and hence increase the value of the fruit. Shatangju is very popular among citrus consumers in China for its superior fruit quality. The main drawback of the cultivar is its short fruit marketing period. Previously, we identified a mutant of Shatangju mandarin , designated as ‘Yuenongwanju’ . Fruit maturation of the MT was significantly delayed by ∼50–60 days compared with that of the WT. Here, by comparing the transcriptomes and the contents of multiple metabolites and two phytohormones of the MT and WT fruits, we provide insight into the role of IAA in citrus fruit maturation.

Consistent with the expression trend of the DEGs involved in the metabolism of ABA and IAA, a gradual increase in the ABA level and a steady decrease in the IAA level were observed in immature fruits of both genotypes. As shown in Figure 6A, an ABA peak was observed at the color change stage in both the MT and the WT fruits. There was no significant difference between the ABA peaks of the two genotypes. However, the ABA contents were unexceptionally lower in all MT fruits at 180, 210, 230 and 250 DPA than in the WT fruits of the same ages. Figure 6A also showed the changes in fruit endogenous free IAA content in both genotypes. It was clear that the fruit free IAA content gradually decreased in both genotypes as the fruit size increased. The IAA content stabilized at a lower level, ∼0.6 ng g−1 FW, after the fruits reached full size. Comparatively, the MT fruits contained significantly higher free IAA than did the WT fruits at all time points prior to 250 DPA. The higher free IAA content in the MT fruit prompted us to investigate whether IAA was responsible for the delayed maturation of the MT fruits. The MG WT fruits, ∼210 days postanthesis, were therefore picked and treated in vitro with IAA in 2019 and 2020 . As expected, the content of free IAA in fruits treated with IAA was significantly higher than that in water-treated and untreated fruits . Treatments with 100 and 1000 μM of IAA increased the pulp IAA levels by ∼15- and 25-fold, respectively, within 24 h. The IAA concentrations in fruits of both treatments decreased rapidly but were still significantly higher than those in the controls after 48 h . It was not unexpected that the IAA-treated WT fruits exhibited a significant delay of color change . Both the untreated CK and the ddH2O-treated fruits completely turned yellow after 25 days of storage at RT. Meanwhile, the fruits under 100 μM IAA treatment had just started changing color, and those under the 1000 μM IAA treatment were still deep green . At 60 days post treatment, fruits treated with 100 μM IAA had almost completely changed color, but those treated with 1000 μM IAA had only slightly changed color .Fruit ripening is controlled by different hormones. For example, blueberry plant pot the ripening of climacteric fruits such as tomato, peach, banana and papaya is regulated by ethylene and IAA . Ripening of non-climacteric fruits, such as strawberry, grape and sweet cherry, is predominantly regulated by ABA and auxin . Auxin, unlike its positive role in enhancing the biosynthesis of ethylene in climacteric fruits , works as a negative regulator, inhibiting ABA biosynthesis in non-climacteric fruits . Citrus fruits are classified as non-climacteric fruits. Their ripening process is therefore considered to be mainly controlled by ABA, although accumulating evidence has suggested that ethylene plays some role, at least in chlorophyll degradation in fruit peel . The role of IAA in citrus fruit ripening has not been extensively explored. In this study, a late ripening mutant of Shatangju mandarin whose fruits mature in February, almost 2 months later than common Shatangju, was characterized. It was found that the younger fruit contained more free IAA and less ABA than did the older fruit, suggesting that IAA might act as a counter maturation agent . Furthermore, exogenous IAA treatment significantly delayed color change in WT fruits . Additionally, significant differences in either IAA or ABA levels existed between the MT and the WT fruits of the same age. That is, the IAA content was higher, whereas the ABA content was lower, in the MT fruit than in the WT fruit. These differences between the two genotypes no longer existed only in fruits that were experiencing color change. Taken together, it seemed that a higher level of free IAA and a lower level of ABA were necessary for maintaining the growth of the citrus fruit until color change. Intriguingly, a match in the IAA or ABA content could always be found between an older MT fruit and a younger WT fruit.

In other words, the MT fruits were able to reach the same physiological status of the WT fruits later , indicating that a slower growth in the early stages of the MT fruit development should be responsible for the lateripening phenotype. Contrary to our findings, a synthetic auxin, NAA, was found to be able to accelerate chlorophyll degradation and carotenoid accumulation in GA- and PDJ-treated Satsuma mandarin fruit . Nevertheless, studies have shown that NAA had bipolar effects on citrus, as manifested by the fact that it either prevents fruitlet drop if used at lower concentrations or promotes fruitlet drop if used at higher concentrations . The fruit of the late-maturing mutant exhibited a significant delay in CB and a dramatic change in the expression of numerous genes. The most pronounced internal changes were that the IAA level was elevated, whereas the ABA level was lowered in growing fruit by the mutation. Correspondingly, some of the IAA and ABA metabolic genes were differentially regulated. Endogenous free IAA levels were determined collectively by the genes of IAA biosynthesis, conjugation and degradation pathways . The indole-3-pyruvate pathway was considered to be the predominant pathway for IAA biosynthesis in higher plants . The rate-limiting step of the pathway that catalyzes the conversion of IPA to IAA is controlled by the YUCCA gene family , and three members of the family, CrYUCCA3, CrYUCCA5 and CrYUCCA8, were found to be expressed differentially between WT and MT . Although the expression of CrYUCCA3 was opposite to that of CrYUCCA8, the three genes combinedly showed a decrease in their expression, which was in agreement with the trend in changes in free auxin. An auxin degradation gene, DAO1, was abundantly, but differentially, expressed between WT and MT, and remarkably, its expression was twice as high in MT as in WT at 230 DPA . It seemed that the expression of a GH3 gene was also elevated in MT fruits . Logically, the elevation in the expression of IAA degradation and conjugation genes should lead to a reduction in the IAA levels in MT fruits, but our findings were the opposite. Multiple studies have shown that alterations in the auxin levels could be corrected by the compensatory changes in auxin metabolism since auxin homeostasis is vital for maintaining normal cell function . For example, Arabidopsis plants lacking AtDAO1 activity did not result in an increase in free IAA levels since the conjugation of IAA was concomitantly elevated . For ABA biosynthesis, the rate-limiting step that cleaves xanthoxin to produce 9-cisviolaxanthin and 9-cis-neoxanthin is controlled by the NCED gene family, and two members of the family, CrNCED3 and CrNCED5, were found to be expressed differentially between WT and MT . Remarkably, the expression level of CrNCED5 in the MT fruit was only half that of the WT fruit at the last two physiological stages, the CB stage and the CC stage . The NCED5 gene was considered to be a key regulator in ABA biosynthesis in citrus fruit . The gene’s expression was closely corelated to the content of ABA in the two studied genotypes . Fruit ripening is a complex biological and physiological process involving changes in many primary and secondary metabolic processes . One of the pronounced changes is the fruit color. Citrus fruit’s color development is correlated with a gradual reduction in green color and a steady increase in mainly orange and yellow colors . Metabolic changes in chlorophylls and carotenoids therefore play a very important role in the coloration of citrus fruit peel . Our RNA-seq data showed that the genes related to chlorophyll and carotenoid metabolisms were differentially expressed. Three genes, CrPSY2, CrZDS1 and CrLCYb2a, which encode three key enzymes in the carotenoid biosynthesis pathway, expressed at higher levels in WT fruit than in MT fruit at 230 and 250 DPA .