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ER-mitochondrial contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer membrane proteins, TRB1, TRB2, and the ER protein, VAP27-1. In Arabidopsis, there are three TraB family isoforms and the trb1/trb2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation, and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TRB1 and TRB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trb1/trb2 and VAP27 mutants ( vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TRB1-regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB family proteins which are component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy. Contact sites permit the exchange of signals and materials between organelles. Here the authors characterize a protein complex associated with an ER-mitochondrial contact site in Arabidopsis that is required for mitochondrial function and mitochondrial turnover

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.

This study compared the physicochemical and functional properties of starches from eight cultivars of avocado seeds. Amylose content, morphology, crystalline structure, swelling power, solubility, thermal and pasting properties, and in vitro digestibility were investigated. The results revealed that the apparent amylose content of starches from avocado seeds varied with different varieties. Light microscopic and scanning electron microscopic examination demonstrated that the eight starches differed slightly in terms of morphology and granule size. The X-ray diffraction and Fourier transform infrared spectroscopy analyses showed that the crystal structure and chemical linkage of the avocado seed starches were similar. However, the pasting, water solubility, and thermal properties of the eight avocado seed starches differed. Importantly, all the starches had high resistant starch content (>60%), with the highest found in Hass seeds (77.83%). To conclude, starch from avocado seeds has a high potential for use in the production of resistant starch.

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.

Background Carotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, ‘Cara cara’ navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation. Results In this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase ( DXS1 ), deoxyxylulose 5-phosphate reductoisomerase ( DXR ), geranylgeranyl diphosphate synthase ( GGPPS2 ), phytoene synthase ( PSY1 ) and lycopene β-cyclase ( LCYB ) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase ( PAL-1 and PAL-4 ), 4-Coumarate-CoA ligase ( 4CL-2 and 4CL-5 ), chalcone synthase ( CHS-1 ) and chalcone isomerase ( CHI ) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed. Conclusions Collectively, our results suggest that DXS1 , DXR , GGPPS2 , PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1 , PAL-4 , 4CL-2 , 4CL-5 , CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

As the most valuable organ of tomato plants, fruit has attracted considerable attention which most focus on its quality formation during the ripening process. A considerable amount of research has reported that fruit quality is affected by metabolic shifts which are under the coordinated regulation of both structural genes and transcriptional regulators. In recent years, with the development of the next generation sequencing, molecular and genetic analysis methods, lots of genes which are involved in the chlorophyll, carotenoid, cell wall, central and secondary metabolism have been identified and confirmed to regulate pigment contents, fruit softening and other aspects of fruit flavor quality. Here, both research concerning the dissection of fruit quality related metabolic changes, the transcriptional and post-translational regulation of these metabolic pathways are reviewed. Furthermore, a weighted gene correlation network analysis of representative genes of fruit quality has been carried out and the potential of the combined application of the gene correlation network analysis, fine-mapping strategies and next generation sequencing to identify novel candidate genes determinants of fruit quality is discussed.

Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

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.

Fruit quality is a very complex trait that is affected by both genetic and non-genetic factors. Generally, low temperature (LT) is used to delay fruit senescence and maintain fruit quality during post-harvest storage but the molecular mechanisms involved are poorly understood. Hirado Buntan Pummelo (HBP;Citrus grandis×C. paradis) fruit were chosen to explore the mechanisms that maintain citrus fruit quality during lengthy LT storage using transcriptome and proteome studies based on digital gene expression (DGE) profiling and two-dimensional gel electrophoresis (2-DE), respectively. Results showed that LT up-regulated stress-responsive genes, arrested signal transduction, and inhibited primary metabolism, secondary metabolism and the transportation of metabolites. Calcineurin B-like protein (CBL)–CBL-interacting protein kinase complexes might be involved in the signal transduction of LT stress, and fruit quality is likely to be regulated by sugar-mediated auxin and abscisic acid (ABA) signalling. Furthermore, ABA was specific to the regulation of citrus fruit senescence and was not involved in the LT stress response. In addition, the accumulation of limonin, nomilin, methanol, and aldehyde, together with the upregulated heat shock proteins, COR15, and cold response-related genes, provided a comprehensive proteomics and transcriptomics view on the coordination of fruit LT stress responses.

How to non-destructively and quickly estimate the storage time of citrus fruit is necessary and urgent for freshness control in the fruit market. As a feasibility study, we present a non-destructive method for storage time prediction of Newhall navel oranges by investigating the characteristics of the rind oil glands in this paper. Through the observation using a digital microscope, the oil glands were divided into three types and the change of their proportions could indicate the rind status as well as the storage time. Images of the rind of the oranges were taken in intervals of 10 days for 40 days, and they were used to train and test the proposed prediction models based on K-Nearest Neighbors (KNN) and deep learning algorithms, respectively. The KNN-based model demonstrated explicit features for storage time prediction based on the gland characteristics and reached a high accuracy of 93.0%, and the deep learning-based model attained an even higher accuracy of 96.0% due to its strong adaptability and robustness. The workflow presented can be readily replicated to develop non-destructive methods to predict the storage time of other types of citrus fruit with similar oil gland characteristics in different storage conditions featuring high efficiency and accuracy.