Response of carbon fixation, allocation, and growth to source-sink manipulation by defoliation in vegetative citrus trees.

Source-sink balance in plants determines carbon distribution, and altering it can impact carbon fixation, transport, and allocation. We aimed to investigate the effect of altered source-sink ratios on carbon fixation, transport, and distribution in 'Valencia' sweet orange (Citrus x sinensis) by various defoliation treatments (0%, 33%, 66%, and 83% leaf removal). Gas exchange parameters were measured on 0 and 10 days after defoliation using A/Ci response curves, and leaf export was measured two days after defoliation using radioisotope tracer techniques. Greater defoliation increased the maximum rate of carboxylation (Vcmax), electron transport rate (J1200), and triose-phosphate utilization rate (TPU). Leaf export was unaffected by defoliation but increased in leaves closer to the shoot apex. Basipetal translocation velocity in the trunk remained unaltered, indicating that more photosynthates remained in the shoot rather than being transported directly to the root sink. Defoliated plants initiated more new flush shoots but accumulated less shoot biomass per plant after 8 weeks. Carbon allocation to fine roots was smaller in defoliated plants, suggesting defoliation led to retention of carbohydrates in aboveground organs such as the trunk and other shoots from previous growing cycles. In conclusion, the low source-sink ratio increased carbon fixation without impacting individual leaf export in citrus. The results suggest that intermediate sinks such as the aboveground perennial organs play a role in mediating the translocation velocity. Further research is necessary to better understand the dynamics of source-sink regulation in citrus trees.

Regulation of magnesium and calcium homeostasis in citrus seedlings under varying magnesium supply.

Magnesium (Mg) and calcium (Ca) are two essential macronutrients in plants; however, the characteristics of Mg and Ca concentrations in organ, subcellular and chemical forms and their relationships in citrus plants, especially under varying Mg supply, are not well understood. In this study, Citrus sinensis seedlings (cv. Xuegan) were cultivated in conditions of Mg deficiency (0 mmol Mg2+ L-1) and Mg sufficiency (2 mmol Mg2+ L-1) to investigate the responses of Mg and Ca homeostasis in different organs and fractions. Compared with Mg sufficiency, Mg deficiency significantly decreased root and shoot growth, with the shoot biomass reduction of branch organs was greater than that of parent organs. In addition to increasing the Ca concentration in the parent stem and lateral root organs, Mg deficiency significantly decreased the concentrations and accumulations of Mg and Ca in citrus seedlings, further altering their distribution in different organs. More than 50% of Ca and Mg were sequestrated in the cell wall and soluble fractions, respectively, with Mg concentration decreasing by 15.4% in roots and 46.9% in leaves under Mg deficiency, while Ca concentration decreased by 27.6% in roots and increased by 23.6% in parent leaves. Approximately 90% of Mg exists in inorganic, water-soluble, and pectate and protein-bound forms, and nearly 90% of Ca exists in water-soluble, pectate and protein-bound, phosphate and oxalate acid forms. Except for the decreased inorganic Mg in roots and water-soluble Mg and Ca in leaves, Mg deficiency increased the proportions of Mg and Ca in all chemical forms. However, Mg deficiency generally increased the Ca/Mg ratio in various organs, subcellular and chemical forms, with negative relationships between Mg concentration and Ca/Mg ratio, and the variations of Mg and Ca were highly separated between Mg supply and organs. In conclusion, our results provide insights into the effects of Mg supply on Mg and Ca homeostasis in citrus plants.

Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in 'Cara cara' navel Orange.

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 ß-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.

Anthocyanin Biosynthesis and DNA Methylation Dynamics in Sweet Orange Fruit [Citrus sinensis L. (Osbeck)] under Cold Stress.

The blood red color of pigmented orange fruit varieties [Citrus sinensis L. (Osbeck)] is due to the presence of anthocyanin pigments that largely contribute to determine the high organoleptic qualities and the nutritional properties of the fruits. The content of pigments in sweet orange depends primarily on genetic factors and on environmental conditions. In particular, it has been extensively shown that cold temperature induces an increase of anthocyanin content that is achieved by the induction of the related gene expression. The purpose of our work is to understand the mechanism underlying the color variegation occurring inside the blood oranges during the cold induction of anthocyanin biosynthesis, despite the fact that the entire fruit is genotypically programmed to produce pigments. Therefore, the amount of anthocyanin and the expression of both structural and regulatory genes have been monitored in either high-pigmented (HP) or not/low pigmented (NP) segments of the same fruit during the storage at 4 °C for a total experimental period of 25 days. Our results clearly indicate that the anthocyanin content is directly correlated with the levels of gene transcription, with higher pigmented areas showing higher enhancement of gene expression. Furthermore, we analyzed the reshaping of the DNA methylation status at the promoter regions of genes related to anthocyanin biosynthetic pathway, such as DFR and Ruby. Our results unequivocally demonstrate that in the promoter regions of both DFR and Ruby, the amount of cytosine methylation strongly decreases along the cold storage in the HP areas, whereas it increases in the NP areas of the same fruit, probably causing a partial block of the gene transcription. Finally, by measuring the changes in the expression levels of the Citrus DNA demethylases, we found that DML1 might play a crucial role in determining the observed demethylation of DFR and Ruby promoters, with its expression induced by cold in the HP areas of the fruits. This is the first report in which different levels of gene expression implicated in anthocyanin production in blood orange fruit is correlated with an epigenetic control mechanism such as promoter methylation.

CisPG21 and CisCEL16 are involved in the regulation of the degradation of cell walls during secretory cavity cell programmed cell death in the fruits of Citrus sinensis (L.) Osbeck.

Pectinase and cellulase participate in cell wall degradation during secretory cavity formation in Citrus fruits. However, it remains unknown how secretory cavity formation is regulated by pectinase and cellulase genes in a schizolysigenous model. Our Results showed that PCD was involved in the schizolysigenous formation of the secretory cavities, and pectinase was involved in the degradation of the middle lamella while pectinase combined with cellulase were responsible for the degradation of the primary cell wall. Furthermore, the expression levels of CisPG21 and CisCEL16 at the intercellular space-forming and lumen-expanding stages with the continuous degradation of the cell wall were significantly higher than those at the initial cell stage and mature stage. The in situ hybridization (ISH) results also showed that CisPG21 and CisCEL16 were mainly located in the degrading cells of secretory cavities, and signals were very strong at the intercellular space-forming and lumen-expanding stages. In conclusion, pectinase and cellulase are directly involved in the degradation of PCD cell walls during schizolysigenous formation in the secretary cavity of Citrus sinensis (L.) Osbeck fruit, while CisPG21 and CisCEL16 are important regulatory genes of pectinase and cellulose during cell wall degradation.

Probing Behavior of Diaphorina citri (Hemiptera: Liviidae) on Valencia Orange Influenced by Sex, Color, and Size.

Candidatus Liberibacter asiaticus Jagoueix, Bové, and Garnier (Rhizobiales: Rhizobiaceae) is transmitted by the psyllid Diaphorina citri Kuwayama and putatively causes Huanglongbing disease in citrus. Huanglongbing has reduced yields by 68% relative to pre-disease yields in Florida. Disease management is partly through vector control. Understanding vector biology is essential in this endeavor. Our goal was to document differences in probing behavior linked to sex. Based on both a literature review and our results, we conclude that there is either no effect of sex or that identifying such an effect requires a sample size at least four times larger than standard methodologies. Including both color and sex in statistical models did not improve model performance. Both sex and color are correlated with body size, and body size has not been considered in previous studies on sex in D. citri in terms of probing behavior. An effect of body size was found wherein larger psyllids took longer to reach ingestion behaviors and larger individuals spent more time-ingesting phloem, but these relationships explained little of the variability in these data. We suggest that the effects of sex can be ignored when running EPG experiments on healthy psyllids.

The Pneumatron: An automated pneumatic apparatus for estimating xylem vulnerability to embolism at high temporal resolution.

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time-consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the "Pneumatron", a device that generates high time-resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r2 = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low-cost, and powerful tool for field measurements, which can significantly improve our understanding of plant-water relations and the mechanisms behind embolism.

A mutant allele of ζ-carotene isomerase (Z-ISO) is associated with the yellow pigmentation of the "Pinalate" sweet orange mutant and reveals new insights into its role in fruit carotenogenesis.

BACKGROUND: Fruit coloration is one of the main quality parameters of Citrus fruit primarily determined by genetic factors. The fruit of ordinary sweet orange (Citrus sinensis) displays a pleasant orange tint due to accumulation of carotenoids, representing ß,ß-xanthophylls more than 80% of the total content. 'Pinalate' is a spontaneous bud mutant, or somatic mutation, derived from sweet orange 'Navelate', characterized by yellow fruits due to elevated proportions of upstream carotenes and reduced ß,ß-xanthophylls, which suggests a biosynthetic blockage at early steps of the carotenoid pathway. RESULTS: To identify the molecular basis of 'Pinalate' yellow fruit, a complete characterization of carotenoids profile together with transcriptional changes in carotenoid biosynthetic genes were performed in mutant and parental fruits during development and ripening. 'Pinalate' fruit showed a distinctive carotenoid profile at all ripening stages, accumulating phytoene, phytofluene and unusual proportions of 9,15,9'-tri-cis- and 9,9'-di-cis-ζ-carotene, while content of downstream carotenoids was significantly decreased. Transcript levels for most of the carotenoid biosynthetic genes showed no alterations in 'Pinalate'; however, the steady-state level mRNA of ζ-carotene isomerase (Z-ISO), which catalyses the conversion of 9,15,9'-tri-cis- to 9,9'-di-cis-ζ-carotene, was significantly reduced both in 'Pinalate' fruit and leaf tissues. Isolation of the 'Pinalate' Z-ISO genomic sequence identified a new allele with a single nucleotide insertion at the second exon, which generates an alternative splicing site that alters Z-ISO transcripts encoding non-functional enzyme. Moreover, functional assays of citrus Z-ISO in E.coli showed that light is able to enhance a non-enzymatic isomerization of tri-cis to di-cis-ζ-carotene, which is in agreement with the partial rescue of mutant phenotype when 'Pinalate' fruits are highly exposed to light during ripening. CONCLUSION: A single nucleotide insertion has been identified in 'Pinalate' Z-ISO gene that results in truncated proteins. This causes a bottleneck in the carotenoid pathway with an unbalanced content of carotenes upstream to ß,ß-xanthophylls in fruit tissues. In chloroplastic tissues, the effects of Z-ISO alteration are mainly manifested as a reduction in total carotenoid content. Taken together, our results indicate that the spontaneous single nucleotide insertion in Z-ISO is the molecular basis of the yellow pigmentation in 'Pinalate' sweet orange and points this isomerase as an essential activity for carotenogenesis in citrus fruits.

Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis.

BACKGROUND: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. RESULTS: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. CONCLUSIONS: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Spatiotemporal association between the mite Brevipalpus yothersi and Citrus leprosis virus C in orange orchards.

Citrus leprosis virus C (CiLV-C) is an economically important pathogen and the main causative agent of leprosis disease in citrus orchards. The main vector of this disease, the mite Brevipalpus yothersi, is widely distributed in Mexican orchards on a wide range of citrus species. Despite the importance of both the virus and the mite, field studies recording their occurrence and co-occurrence are practically non-existent. We systematically sampled orange orchards for both CiLV-C and B. yothersi throughout the year. The distribution of the CiLV-C and B. yothersi was evaluated on each sampling occasion and their spatiotemporal associations were determined. Specifically, 100-112 orange trees, distributed in 18 rows (five or six trees per row), were sampled monthly between March 2017 and February 2018 (11 sampling dates). Twenty leaves per tree were sampled on each occasion. The number of mites per tree and the percentage of leaves per tree with disease symptoms were recorded. On each sampling occasion, spatiotemporal associations between mites and disease were determined using the Spatial Analysis by Distance Indices (SADIE) method. CiLV-C and B. yothersi were identified using molecular methods. Throughout the study, the distribution of CiLV-C was aggregated and the distribution of B. yothersi was random. No association was found between the virus and the mite on any of the sampling dates. In total, 173 mites were collected, but only 43 mites were found to be carrying CiLV-C. The reason for this lack of association between the virus and the mite, as well as the impact of our findings on the epidemiology of the disease in orange orchards, are discussed.

HVA22 from citrus: A small gene family whose some members are involved in plant response to abiotic stress.

The HVA22 gene has been isolated for the first time from the aleurone layer of barley (Hordeum vulgare). Here, we characterized the HVA22 family from citrus (C. clementina and C. sinensis). Twelve genes, 6 in each species, were identified as well as duplication events for some of them. The ORF size ranged from 235 to 804 bp and the protein molecular weight from 94 to 267 kDa. All the citrus HVA22 protein presented transmembrane location and conserved TB2/DP1/HVA22 region. Phylogenetic and gene expression analyses suggested that some citrus HVA22 play a role in flower and fruit development, and that gene expression may be regulated by hormone or environmental conditions. Other regulation levels were also predicted, such as alternative splicing and post-translational modifications. The overall data indicated that citrus HVA22 may be involved in vesicular traffic in stressed cells, and that CcHVA22d could be involved in dehydration tolerance.

A sweet orange mutant impaired in carotenoid biosynthesis and reduced ABA levels results in altered molecular responses along peel ripening.

Citrus fruit ripening is a complex process involving biochemical, physiological and molecular events that differ between the flesh and the peel of the fruit. We characterized sweet orange peel maturation by means of a comparative transcriptomic analysis between Navelate orange (Citrus sinensis L. Osbeck) and its mutant fruit Pinalate, which presents a severe blockage at early steps of the carotenoid biosynthetic pathway and consequently reduced ABA levels. Peel ripening involved the decrease of the photosynthetic activity and the transmembrane transport processes, as well as the buildup of starch and cuticular waxes and the cell wall modification. In addition, a number of biotic and abiotic stress responses, including the defense response, and the response to blue light, water deprivation and abscisic acid stimulus were modulated in a ripening-stage specific manner. The regulation of energy-related processes and secondary metabolism pathways was attenuated in Pinalate, while the molecular mechanisms underlying stress responses displayed dependency on ABA levels. These results indicate that ABA is a key signal inducing stress responses along orange peel ripening, which might determine the fruit postharvest performance.

Responses of reactive oxygen species and methylglyoxal metabolisms to magnesium-deficiency differ greatly among the roots, upper and lower leaves of Citrus sinensis.

BACKGROUND: Magnesium (Mg)-deficiency is one of the most prevalent physiological disorders causing a reduction in Citrus yield and quality. 'Xuegan' (Citrus sinensis) seedlings were irrigated for 16 weeks with nutrient solution containing 2 mM (Mg-sufficiency) or 0 mM (Mg-deficiency) Mg(NO3)2. Thereafter, we investigated the Mg-deficient effects on gas exchange and chlorophyll a fluorescence in the upper and lower leaves, and Mg, reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms in the roots, lower and upper leaves. The specific objectives were to corroborate the hypothesis that the responses of ROS and MG metabolisms to Mg-deficiency were greater in the lower leaves than those in the upper leaves, and different between the leaves and roots. RESULTS: Mg level was higher in the Mg-deficient upper leaves than that in the Mg-deficient lower leaves. This might be responsible for the Mg-deficiency-induced larger alterations of all the measured parameters in the lower leaves than those in the upper leaves, but they showed similar change patterns between the Mg-deficient lower and upper leaves. Accordingly, Mg-deficiency increased greatly their differences between the lower and upper leaves. Most of parameters involved in ROS and MG metabolisms had similar variation trends and degrees between the Mg-deficient lower leaves and roots, but several parameters (namely glutathione S-transferase, sulfite reductase, ascorbate and dehydroascorbate) displayed the opposite variation trends. Obviously, differences existed in the Mg-deficiency-induced alterations of ROS and MG metabolisms between the lower leaves and roots. Although the activities of most antioxidant and sulfur metabolism-related enzymes and glyoxalase I and the level of reduced glutathione in the Mg-deficient leaves and roots and the level of ascorbate in the leaves were kept in higher levels, the levels of malonaldehyde and MG and/or electrolyte leakage were increased in the Mg-deficient lower and upper leaves and roots, especially in the Mg-deficient lower leaves and roots. CONCLUSIONS: The ROS and MG detoxification systems as a whole did not provide sufficient detoxification capacity to prevent the Mg-deficiency-induced production and accumulation of ROS and MG, thus leading to lipid peroxidation and the loss of plasma membrane integrity, especially in the lower leaves and roots.

Rootstock-induced molecular responses associated with drought tolerance in sweet orange as revealed by RNA-Seq.

BACKGROUND: Citrus plants are commercially propagated by grafting, with the rootstock variety influencing a number of horticultural traits, including drought tolerance. Among the different rootstock varieties available for citrus propagation, 'Rangpur' lime is known to confer enhanced tolerance to drought as compared to other citrus rootstocks. The objective of this study was to investigate the poorly understood molecular responses underlying the rootstock-induced drought tolerance in sweet orange. RESULTS: RNA-Seq transcriptome analysis was carried out in leaves of sweet orange grafted on 'Rangpur' lime subjected to control and drought-stress treatments, under greenhouse conditions, using the Illumina HiSeq platform. A total of 41,827 unique transcripts were identified, among which 1764 transcripts showed significant variation (P ≤ 0.001) between the treatments, with 1081 genes induced and 683 repressed by drought-stress treatment. The transcripts were distributed in 44 different categories of cellular component, molecular function and biological process. Several genes related to cell metabolism, including those involved in the metabolisms of cell wall, carbohydrates and antioxidants, light reactions, biotic and abiotic stress responses, as well as genes coding for transcription factors (TFs), protein kinases (PKs) and proteins involved in the abscisic acid (ABA) and ethylene signaling pathways, were differentially regulated by drought stress. RNA-Seq data were validated by quantitative real-time PCR (qPCR) analysis and comparative analysis of expression of the selected genes between sweet orange grafted on drought-tolerant and -sensitive rootstocks revealed new candidate genes for drought tolerance in citrus. CONCLUSIONS: In conclusion, our results showed that only a relatively small but functionally diverse fraction of the sweet orange transcriptome, with functions in metabolism, cellular responses and regulation, was differentially regulated by drought stress. The data suggest that the rootstock-induced drought tolerance in sweet orange includes the transcriptional activation of genes related to the cell wall, soluble carbohydrate and antioxidant metabolisms, biotic and abiotic stress responses, TFs, PKs and ABA signaling pathway, and the downregulation of genes involved in the starch metabolism, light reactions and ethylene signaling. Future efforts to elucidate their functional roles and explore their potential in the citrus genetic improvement should benefit from this data.

Effects of cross-pollination by 'Murcott' tangor on the physicochemical properties, bioactive compounds and antioxidant capacities of 'Qicheng 52' navel orange.

This study investigated the effects of cross-pollination by 'Murcott' tangor on the fruit quality of 'Qicheng52' navel orange, including the physicochemical properties, bioactive compounds and antioxidant capacities. There were no significant differences on the fruit weight, juice yield and pH value of juice between self- and cross-pollinated fruits. However, cross-pollination could significantly improve the fruit quality of 'Qicheng52' fruits by increasing the total soluble solid content from 11.12 ±â€¯1.02 °Brix to 13.86 ±â€¯1.17 °Brix. The results of high performance liquid chromatography analysis of three sugar components indicated that the increase of total sugar was mainly contributed by the increase of fructose and sucrose. Cross-pollination exhibited no effect on the flavonoids content, while the total phenolics content was increased from 210.09 ±â€¯18.55 mg/L to 298.25 ±â€¯29.10 mg/L, which contributed to the higher antioxidant capacity in the cross-pollination fruit juice.

Application of gamma-aminobutyric acid increased the level of phytohormones in Citrus sinensis.

MAIN CONCLUSION: In the current study, we showed that exogenous GABA supplementation increases the endogenous GABA level, several amino acids, and phytohormones in citrus plants, suggesting that GABA works in harmony with phytohormones. Gamma-aminobutyric acid (GABA) plays a key role in cytosolic regulation of pH, controlling of carbon and nitrogen metabolism, and protection against biotic and abiotic stresses. Although it is well-known that GABA is implicated in plant defense and it could act as a signaling molecule, its effect on phytohormones is not completely understood. In this study, we investigated the effect of exogenous GABA on citrus phytohormones using gas chromatography-mass spectrometry. A significant increase in endogenous GABA was observed in GABA-treated plants. The highest increase in GABA was recorded in plants treated with 10 mM 7 days post-treatment. In addition, we observed a moderate increase in several amino acids including glycine, L-alanine, L-proline, L-asparagine, and L-glutamine. The levels of benzoic acid, cinnamic acid, salicylic acid, trans-jasmonic acid, indole acetic acid, indole propionic acid, and abscisic acid were significantly increased in GABA-treated plants compared to the control. The gene expression showed that GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) were induced in GABA-treated plants, indicating a conversion of GABA to succinate. In addition, the gene expression of the regulatory enzymes of the TCA cycle (malate dehydrogenase and succinic dehydrogenase) was upregulated in GABA-treated plants, indicating an induction of respiration. In agreement with the chemical analysis, the gene expression results showed that most of the genes implicated in the biosynthesis of phytohormones were also upregulated in GABA-treated plants. Our results indicated that GABA works in harmony with phytohormones and suggested that regulation of phytohormones by exogenous GABA could play a key role in reducing plant stress.

The transcription factor CsbHLH18 of sweet orange functions in modulation of cold tolerance and homeostasis of reactive oxygen species by regulating the antioxidant gene.

The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest gene families in plants, and participate in various physiological processes, but the physiological role and regulatory function of the majority of bHLHs remain poorly understood. Here, a total of 56 putative CsbHLH genes were identified in sweet orange (Citrus sinensis) based on a genome-wide analysis. The CsbHLH genes, except four members, were distributed throughout nine chromosomes and divided into 19 subgroups. Most of the CsbHLH genes were responsive to cold stress, with the greatest up-regulation being observed in CsbHLH18. CsbHLH18 is localized in the nuclei and has transcriptional activation activity. Overexpression of CsbHLH18 conferred enhanced cold tolerance in transgenic tobacco. The transgenic plants accumulated significantly less reactive oxygen species (ROS), concurrent with increased activities and transcript levels of antioxidant enzymes. In contrast, knockdown of bHLH18 by RNAi in trifoliate orange promoted cold susceptibility, accompanied by down-regulation of antioxidant genes and accumulation of more ROS. Protein-DNA interaction assays demonstrate that CsbHLH18 directly and specifically binds to and activates the promoter of CsPOD. Taken together, these findings indicate that CsbHLH18 plays a positive role in cold tolerance through, at least partly, modulation of ROS homeostasis by directly regulating the antioxidant gene.

Long-term manganese-toxicity-induced alterations of physiology and leaf protein profiles in two Citrus species differing in manganese-tolerance.

Manganese (Mn)-intolerant 'Sour pummelo' (Citrus grandis) and Mn-tolerant 'Xuegan' (Citrus sinensis) seedlings were irrigated for 17 weeks with 2 (control) or 600µM (Mn-toxicity or -excess) MnSO4. C. sinensis had higher Mn-tolerance than C. grandis, as indicated by the higher photosynthesis rates in Mn-excess C. sinensis leaves. Under Mn-toxicity, Mn levels were similar between C. sinensis and C. grandis roots, but lower in C. sinensis leaves than in C. grandis leaves. This might be responsible for C. sinensis Mn-tolerance. Using two-dimensional electrophoresis, we identified more differentially abundant proteins (DAPs) in Mn-excess C. grandis than in Mn-excess C. sinensis leaves, which agrees with the higher Mn levels in Mn-excess C. grandis leaves. DAPs were mainly related to carbohydrate and energy metabolism, stress response, and protein and amino acid metabolism. DAPs involved in the cytoskeleton and signal transduction were found only in Mn-excess C. grandis leaves. We isolated more photosynthesis-related proteins with decreased abundances in Mn-excess C. grandis leaves than in Mn-excess C. sinensis leaves, which might account for the larger decrease in photosynthesis rates in C. grandis leaves. The abundances of proteins involved in reactive oxygen species (ROS) scavenging and photorespiration were increased in Mn-excess C. grandis leaves, while only proteins involved in ROS detoxification were increased in Mn-excess C. sinensis leaves. This agrees with the increased requirement for dissipating the excess absorbed light energy, which was higher in Mn-excess C. grandis leaves than Mn-excess C. sinensis leaves because Mn-toxicity inhibited photosynthesis to a greater degree in C. grandis leaves.

miR3954 is a trigger of phasiRNAs that affects flowering time in citrus.

In plant, a few 22-nt miRNAs direct cleavages of their targets and trigger the biogenesis of phased small interfering RNAs (phasiRNAs) in plant. In this study, we characterized a miRNA triggering phasiRNAs generation, miR3954, and explored its downstream target genes and potential function. Our results demonstrated that miR3954 showed specific expression in the flowers of citrus species, and it targeted a NAC transcription factor (Cs7 g22460) and two non-coding RNA transcripts (lncRNAs, Cs1 g09600 and Cs1 g09635). The production of phasiRNAs was detected from transcripts targeted by miR3954, and was further verified in both sequencing data and transient expression experiments. PhasiRNAs derived from the two lncRNAs targeted not only miR3954-targeted NAC gene but also additional NAC homologous genes. No homologous genes of these two lncRNAs were found in plants other than citrus species, implying that this miR3954-lncRNAs-phasiRNAs-NAC pathway is likely citrus-specific. Transgenic analysis indicated that the miR3954-overexpressing lines showed decreased transcripts of lncRNA, elevated abundance of phasiRNAs and reduced expression of NAC genes. Interestingly, the overexpression of miR3954 leads to early flowering in citrus plants. In summary, our results illustrated a model of the regulatory network of miR3954-lncRNA-phasiRNAs-NAC, which may be functionally involved in flowering in citrus.