TRIPTYCHON-LIKE regulates aspects of both fruit flavor and color in citrus.

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix-loop-helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase, responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator-repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing.

CsMYB96 confers resistance to water loss in citrus fruit by simultaneous regulation of water transport and wax biosynthesis.

A Citrus sinensis R2R3 MYB transcription factor (CsMYB96) has previously been shown to be strongly associated with the expression of many genes related to wax biosynthesis in the fruit. In this study, CsMYB96 was found to alleviate water loss by simultaneously regulating the expression of genes encoding plasma membrane intrinsic proteins (CsPIPs) and wax-related genes. Expression profiling indicated that CsPIP1;1 and CsPIP2;4 had high expression that was representative of other aquaporins, and they were down-regulated in the peel of post-harvest citrus fruit. CsPIP2;4 was further characterized as the predominant CsPIP, with high expression and high-water channel activity. Transient overexpression of CsPIP2;4 accelerated water loss in citrus fruit. In silico analysis further indicated that the expression of CsMYB96 had a significant negative correlation with that of CsPIPs. In vivo and in vitro experiments confirmed that CsMYB96 was able to directly repress the expression of CsPIPs. In addition, CsMYB96 was able to activate wax-related genes and promote wax biosynthesis for defense against water loss. Transient and stable overexpression of CsMYB96 reduced water loss from both citrus fruit and Arabidopsis.

CitWRKY28 and CitNAC029 promote the synthesis of cuticular wax by activating CitKCS gene expression in citrus fruit.

KEY MESSAGE: CitWRKY28 and CitNAC029 are involved in cuticular wax synthesis as indicated by the comparative analysis of fruit aliphatic wax content between Citrus reticulata and Citrus trifoliata and gene co-expression analysis. Cuticular wax covers the fruit surface, playing important roles in reduction of fruit water loss and resistance to pathogen invasion. However, there is limited research on the synthesis and transcriptional regulation of cuticular wax in citrus fruit. In this study, we characterized the variations of aliphatic wax in HJ (Citrus reticulata) and ZK (Citrus trifoliata) from young fruit to mature fruit, as well as performed transcriptome sequencing on 27 samples at different fruit developmental stages. The results revealed that the ZK fruit always had a higher aliphatic wax content than the HJ fruit during development. qRT-PCR analysis demonstrated that two KCS genes, CitKCS1 and CitKCS12, had the most significant difference in expression between HJ and ZK. Furthermore, a heterologous expression assay in Arabidopsis indicated that CitKCS1 and CitKCS12 are involved in cuticular wax synthesis. Subsequently, gene co-expression network analysis screened CitWRKY28 and CitNAC029. Dual luciferase and EMSA assays indicated that CitWRKY28 might bind to the promoter of CitKCS1 and CitKCS12 and CitNAC029 might bind to that of CitKCS1 to activate their expression. Moreover, CitWRKY28 and CitNAC029 could promote the accumulation of cuticular wax in Arabidopsis leaves. Our findings provide new insights into the synthesis and regulation of cuticular wax and valuable information for further mining of wax-related genes in citrus fruit.

Citrus NIP5;1 aquaporin regulates cell membrane water permeability and alters PIPs plasma membrane localization.

KEY MESSAGE: The ER or donut-like structures localized aquaporin NIP5;1, which interacts with PIPs and alters their localization from plasma membrane to donut-like structures, regulates water permeability. NOD26-like intrinsic proteins (NIPs) play important roles in nutrient uptake and response to various stresses. However, there have been few studies of their functions in water transportation in citrus. Here, we demonstrate the functions of a novel citrus NIP aquaporin (CsNIP5;1) via multiple physiological and biochemical experiments. CsNIP5;1 showed high water permeability when expressed in Xenopus laevis oocytes and yeast. However, subcellular localization assays showed that this protein was localized in the endoplasmic reticulum (ER) or donut-like structures in citrus callus and tobacco leaf. Meanwhile, overexpression of CsNIP5;1 led to a reduction in the water permeability of citrus callus. Protein-protein interaction experiments and subcellular localization assays further revealed that CsNIP5;1 physically interacted with PIPs (CsPIP1;1 and AtPIP2;1), which altered their subcellular localization from the plasma membrane to donut-like structures. Together, CsNIP5;1 was identified as a good water channel when expressed in oocytes and yeast. Meanwhile, CsNIP5;1 participated in the regulation of water permeability of citrus callus, which may be associated with CsNIP5;1-induced re-localization of water channels PIPs. In summary, these results provide new insights into the regulatory mechanism of AQPs-mediated water diffusion.

A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata.

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.

An R2R3-MYB transcription factor represses the transformation of α- and ß-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate.

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and ß-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and ß-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of ß-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and ß-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.

Network analysis of postharvest senescence process in citrus fruits revealed by transcriptomic and metabolomic profiling.

Citrus (Citrus spp.), a nonclimacteric fruit, is one of the most important fruit crops in global fruit industry. However, the biological behavior of citrus fruit ripening and postharvest senescence remains unclear. To better understand the senescence process of citrus fruit, we analyzed data sets from commercial microarrays, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry and validated physiological quality detection of four main varieties in the genus Citrus. Network-based approaches of data mining and modeling were used to investigate complex molecular processes in citrus. The Citrus Metabolic Pathway Network and correlation networks were constructed to explore the modules and relationships of the functional genes/metabolites. We found that the different flesh-rind transport of nutrients and water due to the anatomic structural differences among citrus varieties might be an important factor that influences fruit senescence behavior. We then modeled and verified the citrus senescence process. As fruit rind is exposed directly to the environment, which results in energy expenditure in response to biotic and abiotic stresses, nutrients are exported from flesh to rind to maintain the activity of the whole fruit. The depletion of internal substances causes abiotic stresses, which further induces phytohormone reactions, transcription factor regulation, and a series of physiological and biochemical reactions.

CsNIP5;1 acts as a multifunctional regulator to confer water loss tolerance in citrus fruit.

Plant aquaporins facilitate the transport of water across the inner membranes and play an important role in the response to water loss stress. A citrus NOD26-like intrinsic protein, CsNIP5;1, has been investigated to participate in the regulation of water permeability. In the present study, the expression profile indicated that CsNIP5;1 showed high transcription abundance in conducting tissues. Function analysis revealed that CsNIP5;1 reduced water loss of Arabidopsis rosette leaf, as well as promoted the seed germination under hyperosmotic stress. Besides, overexpression of CsNIP5;1 contributed to the alleviation of water loss in citrus fruit and citrus callus during storage. Further metabolomic profiling and RNA-seq analysis of transgenic citrus callus revealed that CsNIP5;1 may modulate the water loss by inducing the accumulation of osmotic adjustment substances and repressing the expression of other AQPs. Moreover, CsWRKY4 and CsWRKY28 were found to directly bind to the promoter and acted as opposite regulators of CsNIP5;1 during the postharvest period. These findings provide new insights into the regulatory mechanism of aquaporins in response to the water loss stress of citrus fruit during postharvest storage.

C24 and C26 aldehydes are potential natural additives of coating for citrus water retention.

Water loss is a key factor for the postharvest senescence of fruit. It has been reported that natural cuticular wax at high concentrations has better performance than commercial coating in water retention of fruit, which can prevent postharvest water loss without the accumulation of off-flavor. Here, we analyzed the correlation between epicuticular wax and postharvest water loss with 75 citrus varieties from a natural population. The water loss rate of the fruit was little influenced by the wax microstructure (stomata and wax crystal morphology), but strongly affected by epicuticular wax components. Further, C24 and C26 aliphatic aldehydes showed the greatest impact on fruit water loss rate, whose correlation coefficients reached -0.63 and -0.67, respectively. These two substances could significantly reduce the fruit water loss rate, indicating that they are potential natural additives to be used in the coating for citrus fruit water retention.

Function and transcriptional regulation of CsKCS20 in the elongation of very-long-chain fatty acids and wax biosynthesis in Citrus sinensis flavedo.

Cuticular wax on plant aerial surfaces plays a vital role in the defense against various stresses, and the genes related to wax metabolism have been well documented in several model plants. However, there is very limited research on the key enzymes and transcription factors (TFs) associated with carbon chain distribution and wax biosynthesis in citrus fruit. In this study, an analysis of wax metabolites indicated that even carbon-chain (C24-C28) metabolites are the dominant wax components in citrus fruit, and a 3-ketoacyl-CoA synthase (KCS) family gene (CsKCS20) plays an important role in the carbon chain distribution during wax biosynthesis in a wax-deficient mutant (MT). Expression of CsKCS20 in yeast indicated that CsKCS20 can catalyze the biosynthesis of C22 and C24 very-long-chain fatty acids (VLCFAs). In addition, transcriptome and sequence analysis indicated that the differential expression of CsKCS20 between the wild-type (WT) and MT fruit can be partly attributed to the regulation of CsMYB96, which was further confirmed by yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays. The functions of CsMYB96 and CsKCS20 in wax biosynthesis were further validated by heterologous expression in Arabidopsis. In summary, this study elucidates the important roles of CsKCS20 and CsMYB96 in regulating VLCFA elongation and cuticular wax biosynthesis, which provides new directions for the improvement of citrus fruit wax quality in genetic breeding programs.

Cytological and proteomic evidence reveals the involvement of mitochondria in hypoxia-induced quality degradation in postharvest citrus fruit.

Hypoxia frequently occurs in postharvest logistics, which greatly influences fruit storability. Here, we for the first time studied the dynamic variations of mitochondrial morphology in living citrus fruit cells, and revealed that waxing treatment-induced hypoxia strongly triggered mitochondrial fission and fragmentation. Correspondingly, hypoxia caused a decline in mitochondrial membrane potential and mobility. Besides, impairment of energetic and redox status was also found in waxed fruit. The proteomic changes of mitochondria after waxing treatment were also characterized. Using weighted gene co-expression network analysis (WGCNA), we identified 167 key hypoxia-responsive proteins, which were mainly involved in fatty acid, amino acid and organic acid metabolism. Metabolite analysis verified that waxing treatment promoted the accumulation of several hypoxic metabolites, such as ethanol, acetaldehyde, succinic acid and γ-aminobutyric acid (GABA). Taken together, our findings provide new insights into the cytological and proteomic responses of mitochondria to hypoxia during fruit storage.

Comprehensive analysis of KCS gene family in Citrinae reveals the involvement of CsKCS2 and CsKCS11 in fruit cuticular wax synthesis at ripening.

Cuticular wax covers the surface of fleshy fruit and plays a protective role in fruit development and postharvest storage, including reducing fruit water loss, resisting biotic and abiotic stress and affecting fruit glossiness. The ß-ketoacyl-CoA synthase (KCS) is the rate-limiting enzyme of very long chain fatty acids (VLCFAs) synthesis, which provides precursors for the synthesis of cuticular wax. In this study, a total of 96 KCS genes were identified in six Citrinae species, including 13, 16, 21, 14, 16 and 16 KCS genes in the primitive species (Atalantia buxifolia), the wild species (Citrus ichangensis), and four cultivated species (Citrus medica, Citrus grandis, Citrus sinensis and Citrus clementina), respectively. Compared with primitive species, wild and cultivated species showed expansion of KCS gene family. Evolutionary analysis of KCS gene family indicated that uneven gain and loss of genes resulted in variable numbers of KCS genes in Citrinae, and KCS genes have undergone purifying selection. Expression profiles in C. sinensis revealed that the KCS genes had diverse expression patterns among various tissues. Furthermore, CsKCS2 and CsKCS11 were predominantly expressed in the flavedo and their expression increased sharply with ripening. Subcellular localization analysis indicated that CsKCS2 and CsKCS11 were located in the endoplasmic reticulum. Further, heterologous expression of CsKCS2 and CsKCS11 in Arabidopsis significantly increased the content of cuticular wax in leaves. Thus, CsKCS2 and CsKCS11 are involved in the accumulation of fruit cuticular wax at ripening. This work will facilitate further functional verification and understanding of the evolution of KCS genes in Citrinae.

Variations of membrane fatty acids and epicuticular wax metabolism in response to oleocellosis in lemon fruit.

Oleocellosis is a physiological disorder causing blemishes on fruit surface. This study investigated the influence of oleocellosis on the membrane fatty acids and wax in lemon fruit rinds at the morphological, physiological, metabolic and molecular levels by using a variety with a high incidence rate of oleocellosis (green lemon). Oleocellosis-damaged rinds showed loose and flaky wax layers with more fissures on the surface, as well as higher contents of C16 and C18 fatty acids and very long chain (VLC) fatty alkanes while lower contents of VLC fatty aldehydes. The main differentially expressed genes, including FabZ, FAD2 and SAD6 involved in the accumulation of C16 and C18 fatty acids and CER1 involved in the transformation of VLC fatty aldehydes to VLC fatty alkanes, were up-regulated by oleocellosis. These results indicate that oleocellosis accelerates the accumulation of membrane free fatty acids and transformation of VLC fatty aldehydes to VLC fatty alkanes.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit.

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

Fatty acid metabolic flux and lipid peroxidation homeostasis maintain the biomembrane stability to improve citrus fruit storage performance.

Little is known about the variations of fresh fruit biomembrane and its physiological and biochemical characteristics during storage. A navel orange mutant 'Gannan No.1' (Citrus sinensis Osbeck) showed higher membrane stability and titratable acid while lower calyx senescence compared with wild-type 'Newhall'. The membrane damage was significantly reduced in 'Gannan No.1' under 10% polyethylene-glycol (41.16% vs. 8.77%) and 30% polyethylene-glycol (52.59% vs.16.11%) treatments on day 45 after harvest. Consistently, membrane electrolyte leakage and malondialdehyde were significantly decreased in 'Gannan No.1', and superoxide dismutase and glutathione reductase were activated. A metabolic analysis was performed to evaluate membrane fatty acid unsaturation and peroxidation. Linolenic acid and hexadecylenic acid contributed to the higher degree of unsaturated fatty acids in 'Gannan No.1'. Furthermore, 'Gannan No.1' accumulated stress-resistant metabolites such as proline, α-tocopherol and glutathione. Correlation analysis of membrane homeostasis indexes with quality parameters showed the importance of biomembrane stability in maintaining citrus fruit quality.

Identification of Key Residues Required for RNA Silencing Suppressor Activity of p23 Protein from a Mild Strain of Citrus Tristeza Virus.

The severe strain of citrus tristeza virus (CTV) causes quick decline of citrus trees. However, the CTV mild strain causes no symptoms and commonly presents in citrus trees. Viral suppressor of RNA silencing (VSR) plays an important role in the successful invasion of viruses into plants. For CTV, VSR has mostly been studied in severe strains. In this study, the N4 mild strain in China was sequenced and found to have high sequence identity with the T30 strain. Furthermore, we verified the functions of three VSRs in the N4 strain, and p23 was found to be the most effective in terms of local silencing suppressor activity among the three CTV VSRs and localized to both nucleus and plasmodesmata, which is similar to CTV T36 strain. Several conserved amino acids were identified in p23. Mutation of E95A/V96A and M99A/L100AA impaired p23 protein stability. Consequently, these two mutants lost most of its suppressor activity and their protein levels could not be rescued by co-expressing p19. Q93A and R143A/E144A abolished p23 suppressor activity only and their protein levels increased to wild type level when co-expressed with p19. This work may facilitate a better understanding of the pathogenic mechanism of CTV mild strains.

Genome of Wild Mandarin and Domestication History of Mandarin.

Mandarin (Citrus reticulata) is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins (MD1 and MD2) in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase (ACO) gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin.