Yunliu Zeng

An integrative analysis of transcriptome and proteome provides new insights into carotenoid biosynthesis and regulation in sweet orange fruits.

An integrative analysis of transcriptome and proteome was performed to identify differential genes/proteins of a red-flesh sweet orange Cara Cara in comparison with a common cultivar Newhall at ripening stages. At the transcript level, gene expression was measured with Massively Parallel Signature Sequencing (MPSS), and 629 genes of these two sweet orange cultivars differed by two fold or more (FDR<0.001). At the protein level, a combination of 2DE and MALDI-TOF-TOF MS identified 48 protein spots differed in relative abundance (P<0.05). The data obtained from comparing transcriptome with proteome showed a poor correlation, suggesting the necessity to integrate both transcriptomic and proteomic approaches in order to get a comprehensive molecular characterization. Function analysis of the differential genes/proteins revealed that a set of candidates was associated with carotenoid biosynthesis and the regulation. Overall, some intriguing genes/proteins were previously unrecognized related with the formation of red-flesh trait, which provided new insights into molecular processes regulating lycopene accumulation in a red-flesh sweet orange. In addition, some genes/proteins were found to be different in expression patterns between the Cara Cara and another red-flesh sweet orange Hong Anliu, and their potential roles were further discussed in the present study.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck]

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

Integration of Metabolomics and Subcellular Organelle Expression Microarray to Increase Understanding the Organic Acid Changes in Post‐harvest Citrus Fruit

Citric acid plays an important role in fresh fruit flavor and its adaptability to post-harvest storage conditions. In order to explore organic acid regulatory mechanisms in post-harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo (HBP; Citrus grandis) fruits. High-performance capillary electrophoresis, subcellular organelle expression microarray, real-time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry (GC-MS), and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC-MS-based metabolic profiling indicated that succinic acid, γ-aminobutyric acid (GABA), and glutamine contents increased, but 2-oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolism processes. In addition, the concentration of organic acids was significantly correlated with senescence-related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post-harvest senescence processes.

Exogenous γ-aminobutyric acid treatment affects citrate and amino acid accumulation to improve fruit quality and storage performance of postharvest citrus fruit

The loss of organic acids during postharvest storage is one of the major factors that reduces the fruit quality and economic value of citrus. Citrate is the most important organic acid in citrus fruits. Molecular evidence has proved that γ-aminobutyric acid (GABA) shunt plays a key role in citrate metabolism. Here, we investigated the effects of exogenous GABA treatment on citrate metabolism and storage quality of postharvest citrus fruit. The content of citrate was significantly increased, which was primarily attributed to the inhibition of the expression of glutamate decarboxylase (GAD). Amino acids, including glutamate, alanine, serine, aspartate and proline, were also increased. Moreover, GABA treatment decreased the fruit rot rate. The activities of antioxidant enzymes and the content of energy source ATP were affected by the treatment. Our results indicate that GABA treatment is a very effective approach for postharvest quality maintenance and improvement of storage performance in citrus production.

Network Analysis of Postharvest Senescence Process in Citrus Fruits Revealed by Transcriptomic and Metabolomic Profiling

The difference in flesh-rind transport of nutrients and water due to the anatomic structural differences among citrus varieties might be an important factor that influences fruit senescence behavior. Citrus (Citrus spp.), a nonclimacteric fruit, is one of the most important fruit crops in global fruit industry. However, the biological behavior of citrus fruit ripening and postharvest senescence remains unclear. To better understand the senescence process of citrus fruit, we analyzed data sets from commercial microarrays, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry and validated physiological quality detection of four main varieties in the genus Citrus. Network-based approaches of data mining and modeling were used to investigate complex molecular processes in citrus. The Citrus Metabolic Pathway Network and correlation networks were constructed to explore the modules and relationships of the functional genes/metabolites. We found that the different flesh-rind transport of nutrients and water due to the anatomic structural differences among citrus varieties might be an important factor that influences fruit senescence behavior. We then modeled and verified the citrus senescence process. As fruit rind is exposed directly to the environment, which results in energy expenditure in response to biotic and abiotic stresses, nutrients are exported from flesh to rind to maintain the activity of the whole fruit. The depletion of internal substances causes abiotic stresses, which further induces phytohormone reactions, transcription factor regulation, and a series of physiological and biochemical reactions.

A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh

Two major chromoplasts are region-specifically formed in citrus and converted from amyloplast precursors. Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast biogenesis, differentiation, and senescence in sweet orange flesh.

Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening

Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post-translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome-wide mapping of in vivo phosphorylation sites in chromoplast-enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide-based affinity chromatography for phosphoprotein enrichment with LC-MS/MS. A total of 109 plastid-localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif-X analysis, two distinct types of phosphorylation sites, one as proline-directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P(3) DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high-level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.

An R2R3‐MYB transcription factor represses the transformation of α‐ and β‐branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate

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.

Distinct Carotenoid and Flavonoid Accumulation in a Spontaneous Mutant of Ponkan (Citrus reticulata Blanco) Results in Yellowish Fruit and Enhanced Postharvest Resistance.

As the most important fresh fruit worldwide, citrus is often subjected to huge postharvest losses caused by abiotic and biotic stresses. As a promising strategy to reduce postharvest losses, enhancing natural defense by potential metabolism reprogramming in citrus mutants has rarely been reported. The yellowish spontaneous mutant of Ponkan (Citrus reticulata Blanco) (YP) was used to investigate the influence of metabolism reprogramming on postharvest performance. Our results show that reduced xanthophyll accumulation is the cause of yellowish coloring of YP and might be attributed to the reduced carotenoid sequestration capacity and upregulated expression of carotenoid cleavage dioxygenase genes. Constantly higher levels of polymethoxylated flavones (PMFs) during the infection and the storage stage might make significant contribution to the more strongly induced resistance against Penicillium digitatum and lower rotting rate. The present study demonstrates the feasibility of applying bud mutants to improve the postharvest performance of citrus fruits.

Salicylic acid treatment reduces the rot of postharvest citrus fruit by inducing the accumulation of H2O2, primary metabolites and lipophilic polymethoxylated flavones.

To comprehensively analyze the effects of salicylic acid (SA) on the storability of Satsuma mandarin (Citrus unshiu), fruits were treated with 2mM SA. The disease incidence of control/SA-treated fruit at 50d and 120d after treatment was 23.3%/10% and 67.3%/23.3%, respectively, suggesting that SA treatment can significantly reduce the rot rate of postharvest citrus fruit. Fruit quality assays revealed that the treatment can maintain fruit firmness without affecting the inner quality. Furthermore, the contents of H2O2 and some defense-related metabolites, such as ornithine and threonine, in citrus pericarp, were significantly increased by SA treatment. Moreover, it was lipophilic polymethoxylated flavones, rather than flavanone glycosides, that accumulated in SA-treated fruits and these can directly inhibit pathogen development. These results suggest that the effects of SA on postharvest citrus fruit may be attributed to the accumulation of H2O2 and defense-related metabolites.