Transcription factor CrWRKY42 coregulates chlorophyll degradation and carotenoid biosynthesis in citrus.

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these 2 processes at the transcriptional level remains largely unknown. In this study, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant "Jinlegan" ([Citrus unshiu × C. sinensis] × C. reticulata) tangor and its wild-type "Shiranui" tangor, which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of ß-carotene hydroxylase 1 (CrBCH1) and activated its expression. The overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene ß-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could bind to the promoters of NONYELLOW COLORING (CrNYC) and STAY-GREEN (CrSGR) and activate their expression, thus promoting chlorophyll degradation. The overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening.

Multiomics integrated with sensory evaluations to identify characteristic aromas and key genes in a novel brown navel orange (Citrus sinensis).

'Zong Cheng' navel orange (ZC) is a brown mutant of Lane Late navel orange (LL) and emits a more pleasant odor than that of LL. However, the key volatile compound of this aroma and underlying mechanism remains unclear. In this study, sensory evaluations and volatile profiling were performed throughout fruit development to identify significant differences in sensory perception and metabolites between LL and ZC. It revealed that the sesquiterpene content varied significantly between ZC and LL. Based on aroma extract dilution and gas chromatography-olfactometry analyses, the volatile compound leading to the background aroma of LL and ZC is d-limonene, the orange note in LL was mainly attributed to octanal, whilst valencene, ß-myrcene, and (E)-ß-ocimene presented balsamic, sweet, and herb notes in ZC. Furthermore, Cs5g12900 and six potential transcription factors were identified as responsible for valencene accumulation in ZC, which is important for enhancing the aroma of ZC.

ß-Cyclocitric acid enhances drought tolerance in peach (Prunus persica) seedlings.

The ß-cyclocitric acid (ß-CCA) is a bioactive apocarotenoid previously shown to improve drought tolerance in annual plants. However, the underlying molecular mechanism of this process remains largely elusive. Moreover, the question about the activity of ß-CCA in perennial fruit crops is still open. Here, we found that treatment of ß-CCA enhances drought tolerance in peach seedlings. The application of ß-CCA significantly increased the relative water content and root activity and reduced the electrolyte leakage of peach seedlings under drought stress. Moreover, treatment with ß-CCA under drought stress increased chlorophyll fluorescence, indicating a positive effect on photosynthesis, while also enhancing superoxide dismutase and peroxidase activity and reducing reactive oxygen species (ROS) levels. Consistent with these alterations, transcriptome analysis revealed an up-regulation of photosynthesis and antioxidant-related genes upon the application of ß-CCA under drought stress. We also detected an induction in genes related to detoxification, environmental adaptation, primary metabolism, phytohormone, phenylpropanoid and the biosynthesis of cutin, suberine and wax, which might contribute to the induction of drought resistance. Altogether, our study reveals that ß-CCA positively modulates peach drought tolerance, which is mainly mediated by enhancing photosynthesis and reducing ROS, indicating the potential of utilizing ß-CCA for drought control in peach and perhaps other fruit crops.

Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in Citrus hesperidium.

Citrus, 1 of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and downregulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene ß-cyclase (CsLCYb2), 2 key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

Two interacting ethylene response factors negatively regulate peach resistance to Lasiodiplodia theobromae.

Gummosis is 1 of the most common and destructive diseases threatening global peach (Prunus persica) production. Our previous studies have revealed that ethylene and methyl jasmonate enhance peach susceptibility to Lasiodiplodia theobromae, a virulent pathogen inducing gummosis; however, the underlying molecular mechanisms remain obscure. Here, 2 ethylene response factors (ERFs), PpERF98 and PpERF1, were identified as negative regulators in peach response to L. theobromae infection. Expression of 2 putative paralogs, PpERF98-1/2, was dramatically induced by ethylene and L. theobromae treatments and accumulated highly in the gummosis-sensitive cultivar. Silencing of PpERF98-1/2 increased salicylic acid (SA) content and pathogenesis-related genes PpPR1 and PpPR2 transcripts, conferring peach resistance to L. theobromae, whereas peach and tomato (Solanum lycopersicum) plants overexpressing either of PpERF98-1/2 showed opposite changes. Also, jasmonic acid markedly accumulated in PpERF98-1/2-silenced plants, but reduction in PpPR3, PpPR4, and PpCHI (Chitinase) transcripts indicated a blocked signaling pathway. PpERF98-1 and 2 were further demonstrated to directly bind the promoters of 2 putative paralogous PpERF1 genes and to activate the ERF branch of the jasmonate/ethylene signaling pathway, thus attenuating SA-dependent defenses. The lesion phenotypes of peach seedlings overexpressing PpERF1-1/2 and PpERF98-1/2 were similar. Furthermore, PpERF98-1/2 formed homodimers/heterodimers and interacted with the 2 PpERF1 proteins to amplify the jasmonate/ethylene signaling pathway, as larger lesions were observed in peach plants cooverexpressing PpERF98 with PpERF1 relative to individual PpERF98 overexpression. Overall, our work deciphers an important regulatory network of ethylene-mediated peach susceptibility to L. theobromae based on a PpERF98-PpERF1 transcriptional cascade, which could be utilized as a potential target for genetic engineering to augment protection against L. theobromae-mediated diseases in crops and trees.

Silencing of PpNRAMP5 improves manganese toxicity tolerance in peach (Prunus persica) seedlings.

The natural resistance-associated macrophage protein (NRAMP) gene family assists in the transport of metal ions in plants. However, the role and underlying physiological mechanism of NRAMP genes under heavy metal toxicity in perennial trees remain to be elucidated. In Prunus persica, five NRAMP family genes were identified and named according to their predicted phylogenetic relationships. The expression profiling analysis indicated that PpNRAMPs were significantly induced by excess manganese (Mn), iron, zinc, and cadmium treatments, suggesting their potential role in heavy metal uptake and transportation. Notably, the expression of PpNRAMP5 was tremendously increased under Mn toxicity stress. Heterologous expression of PpNRAMP5 in yeast cells also confirmed Mn transport. Suppression of PpNRAMP5 through virus-induced gene silencing enhanced Mn tolerance, which was compromised when PpNRAMP5 was overexpressed in peach. The silencing of PpNRAMP5 mitigated Mn toxicity by dramatically reducing Mn contents in roots, and effectively reduced the chlorophyll degradation and improved the photosynthetic apparatus under Mn toxicity stress. Therefore, PpNRAMP5-silenced plants were less damaged by oxidative stress, as signified by lowered H2O2 contents and O2•- staining intensity, also altered the reactive oxygen species (ROS) homeostasis by activating enzymatic antioxidants. Consistently, these physiological changes showed an opposite trend in the PpNRAMP5-overexpressed peach plants. Altogether, our findings suggest that downregulation of PpNRAMP5 markedly reduces the uptake and transportation of Mn, thus activating enzymatic antioxidants to strengthen ROS scavenging capacity and photosynthesis activity, thereby mitigating Mn toxicity in peach plants.

Genome Sequence Resource of Botryosphaeria dothidea Strain XNHG241, a Causal Agent of Peach Gummosis.

Botryosphaeria dothidea is a worldwide pathogenic fungus that causes stem canker, leaf dieback, and fruit rot on a large number of crops and trees. Gummosis caused by B. dothidea is one of the most prevalent and devastating diseases on peach in southern China. This study reported a high-quality and well-annotated genome sequence of B. dothidea strain XNHG241. The findings can be used as a reference for studying fungal biology, pathogenic mechanism of B. dothidea, and the interaction between B. dothidea and host, and eventually facilitate peach gummosis management.

Comprehensive physio-biochemical and transcriptomic characterization to decipher the network of key genes under waterlogging stress and its recuperation in Prunus persica.

Waterlogging is a major abiotic stress that plants encounter as a result of climate change impacts. Peach is very sensitive to hypoxia during waterlogging, which causes poor tree vigor and huge economic losses. The molecular mechanism underlying the peach response to waterlogging and reoxygenation remains unclear. Here, the physiological and molecular responses of 3-week-old peach seedlings under waterlogged and recovery conditions were comprehensively analyzed. As a result, waterlogging significantly reduced plant height and biomass with inhibition of root growth when compared with control and reoxygenation. Similar results were observed for photosynthetic activities and gaseous exchange parameters. Waterlogging increased lipid peroxidation, hydrogen peroxide, proline, glutamic acid and glutathione contents, while superoxide dismutase, peroxidases and catalase activities were decreased. The glucose and fructose contents were accumulated, contrary to sucrose which was reduced remarkably throughout the stress periods. The level of endogenous indole acetic acid (IAA) was increased in waterlogging but decreased after reoxygenation. However, the change trends of jasmonic acid (JA), cytokinins and abscisic acid (ABA) levels were opposite to IAA. In transcriptomic analysis, there were 13,343 differentially expressed genes (DEGs) with higher and 16,112 genes with lower expression. These DEGs were greatly enriched in carbohydrate metabolism, anaerobic fermentation, glutathione metabolism and IAA hormone biosynthesis under waterlogging, while they were significantly enriched in photosynthesis, reactive oxygen species scavenging, ABA and JA hormones biosynthesis in reoxygenation. Moreover, several genes related to stress response, carbohydrate metabolism and hormones biosynthesis were significantly changed in waterlogging and reoxygenation, which indicated unbalanced amino acid, carbon and fatty acid pools in peach roots. Taken together, these results suggest that glutathione, primary sugars and hormone biosynthesis and signaling might play key roles in plant response to waterlogging. Our work provides a comprehensive understanding of gene regulatory networks and metabolites in waterlogging stress and its recuperation, which will facilitate peach waterlogging control.

Integrated metabolite profiling and transcriptome analysis reveal candidate genes involved in the formation of yellow Nelumbo nucifera.

As a worldwide major ornamental flower and a edible plant, lotus (Nelumbo nucifera) is also used as medicine and tea beverage. Here, transcriptome and metabolites of yellow (MLQS) and white (YGB) lotus cultivars during five key flower coloration stages were profiled. 2014 differentially expressed genes were detected with 11 carotenoids in lotus were identified for the first time. Then, regulatory networks between and within functional modules was reconstructed, and the correlation between module-metabolites and gene-metabolites was conducted within 3 core modules. 18 candidate genes related to the formation of yellow flower were screened out and a gene regulatory model for the flower color difference between MLQS and YGB were speculated as follows: The substrate competition between F3'H and F3'5'H and substrate specificity of FLS, together with differential expression of CCD4a and CCD4b were contribute to the differences in flavonoids and carotenoids. Besides, UGT73C2, UGT91C1-2 and SGTase, and regulation of UGTs by transcription factors PLATZ, MADS, NAC031, and MYB308 may also play a role in the upstream regulation. The following verification results indicated that functional differences existed in the coding sequences of NnCCD4b and promoters of NnCCD4a of MLQS and YGB. In all, this study preliminarily reveals the mechanism of yellow flower coloration in lotus and provides new ideas for the study of complex ornamental characters of other plants.

Carotenoid extraction, detection, and analysis in citrus.

Citrus is an important horticultural crop with global, economic and nutritional value. Carotenoids represent the main pigments in citrus fruits and contribute to the esthetic and nutritional value. The complexity of carotenoids in citrus poses a challenge for carotenoid analysis. In this chapter, we describe methods for carotenoid extraction, detection, and analysis that have been optimized for study of citrus fruits. High-performance liquid chromatography (HPLC) and Ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) strategies are used for carotenoid profiling of citrus fruit carotenoids and are explained in detail. We outline the applications, advantages, and disadvantages of using these methods to analyze carotenoids in the main citrus species including mandarin, orange, and pummelo.

Analysis of plant-derived carotenoids in camouflaging stick and leaf insects (Phasmatodea).

A common way to avoid predators is by use of camouflage, a strategy which the stick and leaf insects (Phasmatodea) have refined by appearing as leaves, sticks, lichen, and moss. Stick and leaf insects have perfected their camouflage by sequestering diet-based carotenoids within their exoskeleton. Visual and chemical details of such camouflage have likely been influenced through the millennia of co-evolution between these insects and the plants they mimic. It is this evolutionary struggle that has resulted in a plethora of morphological and chemical adaptations across the stick and leaf insect family tree. In this chapter we discuss prior stick and leaf insect carotenoid studies, proper rearing of specimens, and describe methods for preparation of insect exoskeleton and plant samples, carotenoid extraction and analysis.

Regulation of carotenoid and chlorophyll pools in hesperidia, anatomically unique fruits found only in Citrus.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit "hesperidium" is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus.

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene ß-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening.

Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061.

Chromoplast-specific lycopene ß-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

A Citrus Phosphate Starvation Response Factor CsPHL3 Negatively Regulates Carotenoid Metabolism.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene ß-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

Integrative Physiological, Transcriptional, and Metabolic Analyses Provide Insights Into Response Mechanisms of Prunus persica to Autotoxicity Stress.

Autotoxicity is known as a critical factor in replanting problem that reduces land utilization and creates economic losses. Benzoic acid (BA) is identified as a major autotoxin in peach replant problem, and causes stunted seedling growth or even death. However, the physiological and molecular mechanisms of peach response to BA stress remain elusive. Here, we comprehensively studied the morphophysiological, transcriptional, and metabolic responses of peach plants to BA toxicity. Results showed that BA stress inhibited peach seedlings growth, decreased chlorophyll contents and fluorescence levels, as well as disturbed mineral metabolism. The contents of hydrogen peroxide, superoxide anion, and malondialdehyde, as well as the total antioxidant capacity, were significantly increased under BA stress. A total of 6,319 differentially expressed genes (DEGs) were identified after BA stress, of which the DEGs related to photosynthesis, redox, and ion metabolism were greatly changed; meanwhile, numerous stress-responsive genes (HSPs, GSTs, GR, and ABC transporters) and transcription factors (MYB, AP2/ERF, NAC, bHLH, and WRKY) were noticeably altered under BA stress. BA induced metabolic reprogramming, and 74 differentially accumulated metabolites, including amino acids and derivatives, fatty acids, organic acids, sugars, and sugar alcohols, were identified in BA-stressed roots. Furthermore, an integrated analysis of genes and metabolites indicated that most of the co-mapped KEGG pathways were enriched in amino acid and carbohydrate metabolism, which implied a disturbed carbon and nitrogen metabolism after BA stress. The findings would be insightful in elucidating the mechanisms of plant response to autotoxicity stress, and help guide crops in alleviating replant problem.

Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene ß-cyclase (LCYB) to ß-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene ß-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of ß-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of ß-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

The effect of ß-cyclocitral treatment on the carotenoid content of transgenic Marsh grapefruit (Citrus paradisi Macf.) suspension-cultured cells.

This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase; and the use of this suspension culture to investigate the effects of ß-cyclocitral on carotenoid content and composition. At a ß-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of ß-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following ß-cyclocitral application was ~2-fold higher than the control, reaching 245.8 µg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following ß-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that ß-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.

Metabolite profile comparison of a graft chimera 'Hongrou Huyou' (Citrus changshan-huyou + Citrus unshiu) and its two donor plants.

BACKGROUND: Chimeras synthesized artificially by grafting are crucial to the breeding of perennial woody plants. 'Hongrou Huyou' (Citrus changshan-huyou + Citrus unshiu) is a new graft chimera originating from the junction where a Citrus changshan-huyou ("C") scion was top-grafted onto a stock Satsuma mandarin 'Owari' (C. unshiu, "O"). The chimera was named OCC because the cell layer constitutions were O for Layer 1(L1) and C for L2 and L3. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues in OCC and the two donors were investigated, with the aim of determining the relationship between the layer donors and metabolites. RESULTS: The comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and each of the two donors. The absence or presence of specific metabolites (such as the carotenoids violaxanthin and ß-cryptoxanthin, the volatile hydrocarbon germacrene D, and the primary metabolites citric acid and sorbose) in each tissue was identified in the three phenotypes. According to principal component analysis (PCA), overall, the metabolites in the peel of the chimera were derived from donor C, whereas those in the juice sac of the chimera came from donor O. CONCLUSION: The profiles of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically compared. The content and composition of metabolites were different between the tissues and between OCC and the each of the two donors. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC and was basically consistent with the layer origin; the peel of the chimera was derived from C, and the juice sacs of the chimera came from O. These profiles provide potential chemical markers for genotype differentiation, citrus breeding assessment, and donor selection during artificial chimera synthesis.