Portable Microelectrochemical Sensors for Rapid and Sensitive Determination of Hesperidin in Citrus reticulate 'Chachi' Peel.

Portable and low-cost analytical devices are essential for rapid detection of bioactive substrates in agricultural products. This study presents the first highly integrated microelectrochemical sensor based on pencil graphite for rapid and sensitive detection of hesperidin in Citrus reticulate 'Chachi' peel. The surface morphology and characterization as well as the electrochemical property of pencil graphite was investigated and discussed. A high electrocatalytic efficiency of hesperidin has been found at used pencil graphite-based microelectrodes. Kinetic analysis was carried out to further understand the electrochemical process of hesperidin at a pencil graphite microelectrode. Consequently, a portable and highly-integrated microelectrochemical sensor exhibits a sensitivity of 0.7251 µA cm-2 µM-1 and a detection limit as low as 25 nM (S/N = 3), and high selectivity was fabricated. Proposed microelectrochemical sensors were applied to electrochemically determinate the hesperidin content in the extract of Citrus reticulata "chachi" peel. As a result, the concentration of hesperidin in the actual real sample detected electrochemically with the proposed portable and low-cost microelectrochemical sensors is highly consistent to that obtained with a common chromatographic method, thus indicating the good reliability and that it can be used in practical applications.

Arsenic inhibits citric acid accumulation via downregulating vacuolar proton pump gene expression in citrus fruits.

Citric acid content is a critical quality determinant in citrus (Citrus spp.) fruits. Although arsenic (As) can effectively reduce citric acid content to improve citrus fruit quality, it can have adverse environmental effects. The discovery of nontoxic substitutes is hampered by the incomplete elucidation of the underlying mechanisms of As action in citrus fruits. Metabolic, transcriptomic, and physiological analyses were employed to investigate As action on citric acid accumulation to discover the mechanisms of As action in citrus. The enzyme activity related to citrate biosynthesis was not inhibited and the content of the involved metabolites was not reduced in As-treated fruits. However, the proton pump genes CitPH5 and CitPH1 control the vacuolar citric acid accumulation and transcription factor genes CitTT8 and CitMYB5, which regulate CitPH5 and CitPH1, were downregulated. The oxidative stress-response genes were upregulated in As-treated fruits. The reactive oxygen species (ROS) treatment also downregulated CitTT8 and CitMYB5 in juice cells. The mitochondrial ROS production rate increased in As-treated fruits. AsIII was more potent in stimulating isolated mitochondria to overproduce ROS compared to AsV. Our results indicate that the As inhibition of citric acid accumulation may be primarily due to the transcriptional downregulation of CitPH5, CitPH1, CitTT8, and CitMYB5. As-induced oxidative stress signaling may operate upstream to downregulate these acid regulator genes. Mitochondrial thiol proteins may be the principal targets of As action in citrus fruits.

Metabolic Profiling and Transcriptional Analysis of Carotenoid Accumulation in a Red-Fleshed Mutant of Pummelo (Citrus grandis).

Citrus grandis 'Tomentosa', commonly known as 'Huajuhong' pummelo (HJH), is used in traditional Chinese medicine and can moisten the lungs, resolve phlegm, and relieve coughs. A spontaneous bud mutant, named R-HJH, had a visually attractive phenotype with red albedo tissue and red juice sacs. In this study, the content and composition of carotenoids were investigated and compared between R-HJH and wild-type HJH using HPLC-MS analysis. The total carotenoids in the albedo tissue and juice sacs of R-HJH were 4.03- and 2.89-fold greater than those in HJH, respectively. The massive accumulation of carotenoids, including lycopene, ß-carotene and phytoene, led to the attractive red color of R-HJH. However, the contents of flavones, coumarins and most volatile components (mainly D-limonene and γ-terpinene) were clearly reduced in R-HJH compared with wild-type HJH. To identify the molecular basis of carotenoid accumulation in R-HJH, RNA-Seq transcriptome sequencing was performed. Among 3948 differentially expressed genes (DEGs), the increased upstream synthesis genes (phytoene synthase gene, PSY) and decreased downstream genes (ß-carotene hydroxylase gene, CHYB and carotenoid cleavage dioxygenase gene, CCD7) might be the key factors that account for the high level of carotenoids in R-HJH. These results will be beneficial for determining the molecular mechanism of carotenoid accumulation and metabolism in pummelo.

Integrated transcriptomic and metabolomic analyses reveal key genes controlling flavonoid biosynthesis in Citrus grandis 'Tomentosa' fruits.

As a well-recognized traditional Chinese medicine (TCM), immature fruits of Citrus grandis 'Tomentosa' (CGT) serve to cure chronic cough in humans. Specialized metabolites including flavonoids may have contribute to this curing effect. Knowledge about the molecular mechanisms underlying flavonoid biosynthesis in 'Tomentosa' fruits will, therefore, support the breeding of varieties with improved medicinal properties. Hence, we profiled the transcriptomes and metabolites of the fruits of two contrasting C. grandis varieties, namely 'Zheng-Mao' ('ZM') used in TCM production, and a locally cultivated pomelo, namely 'Guang-Qing' ('GQ'), at four developmental stages. A total of 39 flavonoids, including 14 flavanone/flavone, 5 isoflavonoids, 12 flavonols, and 6 anthocyanins, were identified, and 16 of which were quantitatively determined in the fruits of the two varieties. We found that 'ZM' fruits contain more flavonoids than 'GQ'. Specifically, rhoifolin levels were significantly higher in 'ZM' than in 'GQ'. We annotated 31,510 genes, including 1,387 previously unknown ones, via transcriptome sequencing of 'ZM' and 'GQ.' A total of 646 genes were found to be differentially expressed between 'ZM' and 'GQ' throughout at all four fruit developmental stages, indicating that they are robust expression markers for future breeding programs. Weighted gene co-expression network analysis identified 18 modules. Combined transcriptional and metabolic analysis revealed 25 genes related to flavonoid biosynthesis and 16 transcriptional regulators (MYBs, bHLHs, WD40) that may be involved in the flavonoids biosynthesis in C. grandis 'Tomentosa' fruits.

Chemical Composition of Cuticle and Barrier Properties to Transpiration in the Fruit of Clausena lansium (Lour.) Skeels.

The plant cuticle, as a lipid membrane covering aerial plant surfaces, functions primarily against uncontrolled water loss. Herein, the cuticle chemical composition and the transpiration of wampee fruit (Clausena lansium (Lour.) Skeels) at the green, turning, and yellow stages in cultivars of "Jixin" and "Tianhuangpi" were comprehensively studied. The coverage of wax and cutin monomers per unit of fruit surface area at the green stage was lower in "Jixin" than in "Tianhuangpi" and increased gradually during development. Cutin monomers accumulated ranging from 22.5 µg cm-2 (green) to 52.5 µg cm-2 (turning) in "Jixin" and from 36.5 µg cm-2 (green) to 81.7 µg cm-2 (yellow) in "Tianhuangpi." The total composition of waxes ranged between 6.0 µg cm-2 (green) and 11.1 µg cm-2 (turning) in "Jixin," while they increased from 7.4 µg cm-2 (green) to 16.7 µg cm-2 (yellow) in "Tianhuangpi." Cutin monomers were dominated by ω-, mid-dihydroxy fatty acids (over 40%), followed by multiple monomers of α,ω-dicarboxylic acids with or without added groups, α-monocarboxylic acids with or without ω- or mid-chain hydroxy or mid-epoxy groups, primary alcohols, and phenolics. The very-long-chain (VLC) aliphatic pattern of cuticular waxes was prominently composed of n-alkanes (ranging from 21.4% to 39.3% of total wax content), fatty acids, primary alcohols, and aldehydes. The cyclic waxes were dominated by triterpenoids (between 23.9 and 51.2%), sterols, and phenolics. Water loss in wampee fruit exhibited linear changes over time, indicating an overall monofunctional barrier to transpiration. Permeance for water in wampee fruit was higher at the green stage than at the yellow stage in both "Jixin" and "Tianhuangpi," which showed a negative correlation with the changes of VLC n-alkanes. The results showed the cuticular chemicals, including cutin monomers and waxes, in wampee fruit and further indicated the potential contributions of the cuticular chemical composition to the physiological functions in fruits.

Changes of Morphology, Chemical Compositions, and the Biosynthesis Regulations of Cuticle in Response to Chilling Injury of Banana Fruit During Storage.

The plant cuticle covers almost all the outermost surface of aerial plant organs, which play a primary function in limiting water loss and responding to the environmental interactions. Banana fruit is susceptible to thermal changes with chilling injury below 13°C and green ripening over 25°C. Herein, the changes of surface morphology, chemical compositions of cuticle, and the relative expression of cuticle biosynthesis genes in banana fruit under low-temperature storage were investigated. Banana fruit exhibited chilling injury rapidly with browned peel appearance stored at 4°C for 6 days. The surface altered apparently from the clear plateau with micro-crystals to smooth appearance. As compared to normal ones, the overall coverage of the main cuticle pattern of waxes and cutin monomers increased about 22% and 35%, respectively, in browned banana stored under low temperature at 6 days. Fatty acids (C16-C18) and ω-OH, mid-chain-epoxy fatty acids (C18) dominated cutin monomers. The monomers of fatty acids, the low abundant ω, mid-chain-diOH fatty acids, and 2-hydroxy fatty acids increased remarkably under low temperature. The cuticular waxes were dominated by fatty acids (> C19), n-alkanes, and triterpenoids; and the fatty acids and aldehydes were shifted to increase accompanied by the chilling injury. Furthermore, RNA-seq highlighted 111 cuticle-related genes involved in fatty acid elongation, biosynthesis of very-long-chain (VLC) aliphatics, triterpenoids, and cutin monomers, and lipid-transfer proteins were significantly differentially regulated by low temperature in banana. Results obtained indicate that the cuticle covering on the fruit surface was also involved to respond to the chilling injury of banana fruit after harvest. These findings provide useful insights to link the cuticle on the basis of morphology, chemical composition changes, and their biosynthesis regulations in response to the thermal stress of fruit during storage.

Activation of RAW264.7 macrophages by an acidic polysaccharide derived from Citrus grandis 'Tomentosa'.

Citrus grandis 'Tomentosa' which is a special Citrus cultivar, has been employed as cough suppressant and expectorant in traditional Chinese medicine for thousands of years. The aim of this study is to investigate the immunomodulatory role of an acidic polysaccharide (designated as CGTP-AP) purified from C. grandis 'Tomentosa'. CGTP-AP showed effective immune activation in RAW264.7 macrophages at the concentration of 1-100 µg/mL. CGTP-AP could promote the release of NO in dose- and time-dependent manners. Enzyme-Linked Immunosorbent Assay (ELISA) and RT-PCR analysis demonstrated that CGTP-AP could stimulate the secretion of TNF-α and IL-6 in a dosage-dependent way. Western blot analysis and RT-PCR analysis indicated that CGTP-AP treatment could induce the iNOS and COX-2 expression in RAW264.7 macrophages. By conducting the inhibitors experiments, the activation of NF-κB and MAPK signaling pathways by CGTP-AP treatment was confirmed. Therefore, the present results declared that CGTP-AP could be a promising candidate as a potent immunomodulator for the application in future pharmaceutical development.

A comparative UPLC-Q-Orbitrap-MS untargeted metabolomics investigation of different parts of Clausena lansium (Lour.) Skeels.

In this study, the non-targeted large-scale plant metabolomics (UPLC-Q-Orbitrap-MS) was performed for the comparison of chemical profiling of the leaves, barks, flowers, peels, pulps, and seeds of Clausena lansium (Lour.) Skeels (called "wampee"). A total of 364 metabolites were identified, and 62 potential biomarkers were selected by the multivariate statistical analysis. Hierarchical cluster analysis suggested that the selected biomarkers were significant differential metabolites among various parts of wampee. Metabolic pathway analysis showed a significant enrichment of the "Flavone and flavonol synthesis" and "Isoquinoline alkaloid biosynthesis" pathway. This study provides important information for the isolation and identification of functional components from different tissues of wampee and the metabolic biosynthesis pathway elucidation in detail.

Structural elucidation of an acidic polysaccharide from Citrus grandis 'Tomentosa' and its anti-proliferative effects on LOVO and SW620 cells.

Citrus grandis 'Tomentosa' (CGT) which is a region-famous medicinal and edible plant contains plentiful bioactive polysaccharides, however, its chemical structures and specific bioactivities still need to be further explored. In the present study, an acidic polysaccharide (CGTP-AP) was extracted and purified from the pulps of CGT. The structure elucidation and anti-cancer activity of CGTP-AP were investigated. Structure characterization indicated that CGTP-AP was a homogeneous heteropolysaccharide composed of arabinose, galactose and galacturonic acid in a molar ratio of 2.45:1:2.77, with an average molecular weight of 2721.68 kDa. Partial acid hydrolysis, methylation and NMR spectrometry revealed that the backbone of CGTP-AP mainly composed of (1 → 4)-α-D-galacturonan, while the branch principally consisted of (1 → 5)-α-L-Araf. In addition, CGTP-AP exhibited effective anti-proliferation against colon cancer cells LOVO and SW620 cells in dose- and time-dependent manners, with IC50 values of 5.55, 4.35 and 3.52 mg mL-1 after 24, 48 and 72 h, and 5.33, 3.63 and 2.97 mg mL-1 after 24, 48 and 72 h, respectively. This study indicated that CGTP-AP might be utilized as a promising food supplement for the patients of colon disorders.

Aromatic dipeptide Trp-Ala can be transported by Arabidopsis peptide transporters AtPTR1 and AtPTR5.

Dipeptides with an aromatic residue at the N-terminal position induced lower inward currents or blocked leak currents in Xenopus oocytes expressing the proton-coupled peptide transporter AtPTR1 or AtPTR5 of Arabidopsis thaliana compared with dipeptides with an aromatic residue at the C-terminal position. Here, AtPTR1 and AtPTR5 were expressed in a yeast mutant of peptide transporter (ptr2) with tryptophan auxotrophy. Growth assays showed that Trp-Ala could be transported by both AtPTR1 and AtPTR5 as efficiently as Ala-Trp. Our data suggested that the previous finding in Xenopus oocytes might be an artifact of heterologous expression, and that AtPTR1 and AtPTR5 expressed in yeast could transport dipeptides with an aromatic residue at the N-terminal position.

Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply.

The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.