[Identification of CjSPL gene family in Ziyang Xiangcheng rootstock and expression pattern analysis].

Squamosa promoter binding protein-like (SPL) family is a group of important transcription factors involved in the regulation of plant growth and development and the response to environmental stress, but there are few studies in perennial fruit trees such as citrus. In this study, Ziyang Xiangcheng (Citrus junos Sib.ex Tanaka), an important rootstock of Citrus, was used as the material for analysis. Based on plantTFDB transcription factor database and sweet orange genome database, 15 SPL family members were genome-widely identified and cloned from Ziyang Xiangcheng, and named CjSPL1-CjSPL15. Sequence analysis showed that the open reading frame (ORF) length of CjSPLs ranged from 393 bp to 2 865 bp, encoding 130-954 amino acids. Phylogenetic tree divided 15 CjSPLs into 9 subfamilies. Gene structure and conserved domain analysis predicted 20 different conserved motifs and SBP basic domains. Analysis of cis-acting promoter elements predicted 20 different promoter elements, including those related to plant growth and development, abiotic stress and secondary metabolites. The expression patterns of CjSPLs under drought, salt and low temperature stresses were analyzed by real-time fluorescence quantitative PCR (qRT-PCR), and many CjSPLs were significantly up-regulated after stress treatment. This study provides a reference for further study on the function of SPL family transcription factors in citrus and other fruit trees.

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.

Effect of the Rare Earth Element Lanthanum (La) on the Growth and Development of Citrus Rootstock Seedlings.

Rare earth elements (REEs) can affect the growth and development of plants. However, few studies have been carried out on the effects of REEs on citrus seedlings. In this study, the growth parameters, toxicity symptoms, chlorophyll content, and La content of three citrus rootstocks are analyzed under different concentrations of La, a representative REE. The results show that the growth of citrus rootstock seedlings was stimulated at La ≤ 0.5 mmol·L-1 and inhibited at concentrations above 1 mmol·L-1. The chlorophyll and carotenoid contents of trifoliate orange (Poncirus trifoliata L. Raf.) and Ziyang Xiangcheng (C. junos Sieb. ex Tanaka) leaves of plants grown at low concentrations of La (≤1.5 mmol·L-1) were similar to those of the control but were significantly reduced at 4 mmol·L-1 La. Toxic symptoms gradually appeared with increasing La concentrations, with yellowed leaves and burst veins appearing at 4 mmol·L-1 La. The symptoms of toxicity were most severe in trifoliate orange, followed by Shatian Pomelo (Citrus grandis var. shatinyu Hort) and then Ziyang Xiangcheng. Moreover, in leaves, the Ca content was significantly negatively correlated with La content (p < 0.01). These results indicate that La has a hormesis effect on the growth of citrus rootstocks. Of the studied citrus seedlings, Ziyang Xiangcheng is the most resistant to La.

Lipidomic and transcriptomic analysis reveals reallocation of carbon flux from cuticular wax into plastid membrane lipids in a glossy "Newhall" navel orange mutant.

Both cuticle and membrane lipids play essential roles in quality maintenance and disease resistance in fresh fruits. Many reports have indicated the modification of alternative branch pathways in epicuticular wax mutants; however, the specific alterations concerning lipids have not been clarified thus far. Here, we conducted a comprehensive, time-resolved lipidomic, and transcriptomic analysis on the "Newhall" navel orange (WT) and its glossy mutant (MT) "Gannan No. 1". The results revealed severely suppressed wax formation accompanied by significantly elevated production of 36-carbon plastid lipids with increasing fruit maturation in MT. Transcriptomics analysis further identified a series of key functional enzymes and transcription factors putatively involved in the biosynthesis pathways of wax and membrane lipids. Moreover, the high accumulation of jasmonic acid (JA) in MT was possibly due to the need to maintain plastid lipid homeostasis, as the expression levels of two significantly upregulated lipases (CsDAD1 and CsDALL2) were positively correlated with plastid lipids and characterized to hydrolyze plastid lipids to increase the JA content. Our results will provide new insights into the molecular mechanisms underlying the natural variation of plant lipids to lay a foundation for the quality improvement of citrus fruit.

Systematic analysis of the basic/helix-loop-helix (bHLH) transcription factor family in pummelo (Citrus grandis) and identification of the key members involved in the response to iron deficiency.

BACKGROUND: Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown. RESULTS: In this study, we identified a total of 128 CgbHLHs from pummelo (Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency. CONCLUSIONS: In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of CgbHLH genes in citrus.

Expression of a Chromoplast-Specific Lycopene ß-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat.

Carotenoids are the principal pigments in the loquat. Although the metabolic pathway of plant carotenoids has been extensively investigated, few studies have been explored the regulatory mechanisms of loquat carotenoids because knowledge of the loquat genome is incomplete. The chromoplast-specific lycopene ß-cyclase gene (CYC-B) could catalyze cyclization of lycopene to ß-carotene. In this study, the differential accumulation patterns of loquat with different colors were analyzed and virus-induced gene silencing (VIGS) was utilized in order to verify CYC-B gene function. Using a cloning strategy of homologous genes, a CYC-B gene orthologue was successfully identified from the loquat. At a later stage of maturation, CYC-B gene expression and carotenoids concentrations in the 'Dawuxing' variety were higher than in 'Chuannong 1-5-9', possibly leading to the difference in pulp coloration of loquat. Interference of CYC-B gene expression in the loquat demonstrated clear visual changes. The green color in negative control fruits became yellow, while TRV2-CYC-B silenced fruits remained green. CYC-B gene expression and total carotenoid content in the pulp decreased by 32.5% and 44.1%, respectively. Furthermore, multiple key genes in the carotenoid metabolic pathway synergistically responded to downregulation of CYC-B gene expression. In summary, we provide direct evidences that CYC-B gene is involved in carotenoid accumulation and coloration in the loquat.

Whole-transcriptome RNA sequencing reveals the global molecular responses and ceRNA regulatory network of mRNAs, lncRNAs, miRNAs and circRNAs in response to copper toxicity in Ziyang Xiangcheng (Citrus junos Sieb. Ex Tanaka).

BACKGROUND: Copper (Cu) toxicity has become a potential threat for citrus production, but little is known about related mechanisms. This study aims to uncover the global landscape of mRNAs, long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in response to Cu toxicity so as to construct a regulatory network of competing endogenous RNAs (ceRNAs) and to provide valuable knowledge pertinent to Cu response in citrus. RESULTS: Tolerance of four commonly used rootstocks to Cu toxicity was evaluated, and 'Ziyang Xiangcheng' (Citrus junos) was found to be the most tolerant genotype. Then the roots and leaves sampled from 'Ziyang Xiangcheng' with or without Cu treatment were used for whole-transcriptome sequencing. In total, 5734 and 222 mRNAs, 164 and 5 lncRNAs, 45 and 17 circRNAs, and 147 and 130 miRNAs were identified to be differentially expressed (DE) in Cu-treated roots and leaves, respectively, in comparison with the control. Gene ontology enrichment analysis showed that most of the DEmRNAs and targets of DElncRNAs and DEmiRNAs were annotated to the categories of 'oxidation-reduction', 'phosphorylation', 'membrane', and 'ion binding'. The ceRNA network was then constructed with the predicted pairs of DEmRNAs-DEmiRNAs and DElncRNAs-DEmiRNAs, which further revealed regulatory roles of these DERNAs in Cu toxicity. CONCLUSIONS: A large number of mRNAs, lncRNAs, circRNAs, and miRNAs in 'Ziyang Xiangcheng' were altered in response to Cu toxicity, which may play crucial roles in mitigation of Cu toxicity through the ceRNA regulatory network in this Cu-tolerant rootstock.

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.

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.

Metabolic Profiling of Pitaya (Hylocereus polyrhizus) during Fruit Development and Maturation.

Pitaya (Hylocereus polyrhizus) has attracted much interest from consumers as it is a novelty fruit with high nutrient content and a tolerance to drought stress. As a group of attractive pigment- and health-promoting natural compounds, betalains represent a visual feature for pitaya fruit quality. However, little information on the correlation between betalains and relevant metabolites exists so far. Currently, color (Commission International del'Eclairage, CIE) parameters, betalain contents, and untargeted metabolic profiling (gas chromatography-time-of-flight-mass spectrometry, GC⁻MS and liquid chromatography tandem mass spectrometry, LC⁻MS) have been examined on 'Zihonglong' fruits at nine different developmental stages, and the variation character of the metabolite contents was simultaneously investigated between peel and pulp. Furthermore, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to explore metabolite profiles from the fruit samples. Our results demonstrated that the decrease of amino acid, accompanied by the increase of sugars and organic acid, might contribute to the formation of betalains. Notably, as one of four potential biomarker metabolites, citramalic acid might be related to betalain formation.

Integrated transcriptomic and metabolomic analyses of a wax deficient citrus mutant exhibiting jasmonic acid-mediated defense against fungal pathogens.

Naturally, resistant crop germplasms are important resources for managing the issues of agricultural product safety and environment deterioration. We found a spontaneous mutant of 'Newhall' navel orange (Citrus sinensis Osbeck) (MT) with broad-spectrum protections against fungal pathogens in the orchard, postharvest-storage, and artificial inoculation conditions. To understand the defense mechanism of MT fruit, we constructed a genome-scale metabolic network that integrated metabolome and transcriptome datasets. The coordinated transcriptomic and metabolic data were enriched in two sub-networks, showing the decrease in very long chain fatty acid (by 41.53%) and cuticular wax synthesis (by 81.34%), and increase in the synthesis of jasmonic acid (JA) (by 95.23%) and JA-induced metabolites such as 5-dimethylnobietin (by 28.37%) in MT. Furthermore, cytological and biochemical analyses confirmed that the response to fungal infection in MT was independent of wax deficiency and was correlated with the levels of jasmonates, and the expression of plant defensin gene PDF1.2. Results of exogenous application of MeJA and JA inhibitors such as propyl gallate proved that JA-mediated defense contributes to the strong tolerance against pathogens in MT. Our results indicated that jasmonate biosynthesis and signaling are stimulated by the fatty acid redirection of MT, and participate in the tolerance of pathogenic fungi.

Regulation of cuticle formation during fruit development and ripening in 'Newhall' navel orange (Citrus sinensis Osbeck) revealed by transcriptomic and metabolomic profiling.

Fruit cuticle, which is composed of cutin and wax and biosynthesized during fruit development, plays important roles in the prevention of water loss and the resistance to pathogen infection during fruit development and postharvest storage. However, the key factors and mechanisms regarding the cuticle biosynthesis in citrus fruits are still unclear. Here, fruit cuticle of 'Newhall' navel orange (Citrus sinensis Osbeck) was studied from the stage of fruit expansion to postharvest storage from the perspectives of morphology, transcription and metabolism. The results demonstrated that cutin accumulation is synchronous with fruit expansion, while wax synthesis is synchronous with fruit maturation. Metabolic profile of fruits peel revealed that transition of metabolism of fruit peel occurred from 120 to 150 DAF and ABA was predicted to regulate citrus wax synthesis during the development of Newhall fruits. RNA-seq analysis of the peel from the above two stages manifested that the genes involved in photosynthesis were repressed, while the genes involved in the biosynthesis of wax, cutin and lignin were significantly induced at later stages. Further real-time PCR predicted that MYB transcription factor GL1-like regulates citrus fruits wax synthesis. These results are valuable for improving the fruit quality during development and storage.