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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 β-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and β-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 β-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and β-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.

Key messageCitWRKY28 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.

Key messageThe 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.

Temperature pretreatment is one of the most important factors which significantly affects the postharvest quality of citrus fruit. In this study, late-ripening Valencia orange (citrus sinensis) fruits were used to investigate the effect of short-term treatment at low (6°C), room (20°C), and high (40°C) temperatures on fruit quality. Our results revealed that both low and room-temperature treatments maintained the content of sugars and organic acids, whereas high-temperature treatments elevated the accumulation of sugars but decreased the content of citric acid. In fruit peel (flavedo and albedo), the accumulation of sugars and organic acids responding to temperatures was diverse and mostly different from that in the pulp. Meanwhile, GABA and several amino acids were upregulated under short-term high-temperature treatment but downregulated in response to low-temperature treatment in both peel and pulp. Furthermore, PCA and correlation analysis revealed that the short-term temperature treatments changed the metabolic flow, and GABA was positively correlated with sugars and organic acids. Our study analyzed the metabolic changes of fruit peel and pulp in response to short-term temperature treatments and revealed that GABA may act as a signaling molecular involved in temperature-controlled quality changes.

This study investigated the effects of Ethephon (CEPA) and Gibberellic acid (GA3) treatments on the post-harvest flavor quality of ‘Yunning No. 1’ green lemon. A comprehensive analysis was conducted on the changes in primary metabolites (sugars, organic acids, amino acids, alcohols) in the pulp and peel, as well as those in major volatile compounds in the peel during fruit storage. The results showed that CEPA treatment initially increased volatile compounds like monoterpenes and sesquiterpenes in the fruit peel during early storage, but later decreased these compounds along with total sugar and amino acid content in the pulp. Conversely, GA3 treatment markedly delayed the decline in sugars and organic acids in the fruit peel, preserved the amino acid content in the pulp and the alcohol content in the peel, and delayed the decrease in volatile compound content in the peel. In conclusion, GA3 treatment effectively delayed the decline in primary metabolites and volatile compounds to maintain the storage quality of green lemon; therefore, GA3 represents a suitable strategy for the preservation of green lemons. CEPA temporarily improved aroma but accelerated quality deterioration, making it better suited for short-term degreening. This study offers a theoretical foundation for optimizing post-harvest degreening and preservation techniques of green lemons.

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.

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.

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 β‐branch carotenoid metabolism. In this study, an R2R3‐MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α‐ and β‐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 β‐carotene hydroxylases 2 ( CrBCH2 ) and 9‐cis‐epoxycarotenoid dioxygenase 5 ( CrNCED5 ) was responsible for the delay in the transformation of α‐ and β‐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.

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 genusCitrus. 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 thenmodeled 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.

The traditional technology for internal quality detection of citrus fruit is believed to be destructive, inefficient, and error-prone. To address these issues, a novel method has been developed to evaluate nondestructively the sugar-acid ratio (SAR) of soluble solids by using the oil glands density (OGD). In this study, a total of 584 samples with different widths were collected. The sample data were correlated that the SAR and OGD decreased with time during storage. The relevance with time was significant between SAR and OGD of citrus positively correlated in the calibration group (i?2=0.82), which indicated that OGD could be used to predict the internal quality of citrus. It indicated that a new generation of digital microscopy could provide an alternative to predict nondestructively the internal quality of citrus fruit, as well as some theoretical insight for further studies of online quality detection in the future.