Analytical strategies to identify multicomponent quality markers for commercial Hua-ju-hong using multidimensional chemical analysis.

Hua-ju-hong (HJH) is a Chinese medicinal material obtained from Citrus grandis 'Tomentosa' (CGT) and Citrus grandis (L.) Osbeck (CG) with various commercial specifications. It is known for relieving cough and dispelling phlegm. To reveal the quality marker for distinguishing the various HJH, 215 batches of commercial HJH were studied systematically using multidimensional chemical analysis. Ten major components were identified by high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and quantified via high-performance liquid chromatography coupled with diode array detection. In this study, a rapid, efficient, and low-cost chromatographic method was established. Total coumarin-hemiterpene and total coumarin-monoterpene were first classified and analyzed in HJH. The result indicated that the main component, naringin, was not the quality marker for differentiating CGT from CG. For reflecting the unique medicinal and food value of HJH, coumarins should be the more potential quality markers. Flavonoids were the possible quality markers for distinguishing two growth stages of fruit-exocarp and young fruit. For the first time, two chemotypes of HJH were identified in CG. This study provides a convenient yet reliant chromatographic method and novel yet systematic strategies for overall quality control of commercial HJH.

Shatianyu dietary fiber (Citrus grandis L. Osbeck) promotes the production of active metabolites from its flavonoids during in vitro colonic fermentation.

BACKGROUND: Recent studies reveal that dietary fiber (DF) might play a critical role in the metabolism and bioactivity of flavonoids by regulating gut microbiota. We previously found that Shatianyu (Citrus grandis L. Osbeck) pulp was rich in flavonoids and DF, and Shatianyu pulp flavonoid extracts (SPFEs) were dominated by melitidin, obviously different from other citrus flavonoids dominated by naringin. The effects of Shatianyu pulp DF (SPDF) on the microbial metabolism and bioactivity of SPFEs is unknown. RESULTS: An in vitro colonic fermentation model was used to explore the effects of SPDF on the microbial metabolism and antioxidant activity of SPFEs in the present study. At the beginning of fermentation, SPDF promoted the microbial degradation of SPFEs. After 24 h-fermentation, the supplemented SPFEs were almost all degraded in SPFEs group, and the main metabolites detected were the dehydrogenation, hydroxylation and acetylation products of naringenin, the aglycone of the major SPFEs components. However, when SPFEs fermented with SPDF for 24 h, 60.7% of flavonoid compounds were retained, and SPFEs were mainly transformed to the ring fission metabolites, such as 3-(4-hydroxyphenyl) propionic acid, 3-phenylpropionic acid and 3-(3-hydroxy-phenyl) propionic acid. The fermentation metabolites of SPFEs showed stronger antioxidant activity than the original ones, with a further increase in SPDF supplemented group. Furthermore, SPFEs enriched microbiota participating in the deglycosylation and dehydrogenation of flavonoids, while co-supplementation of SPDF and SPFEs witnessed the bloom of Lactobacillaceae and Lactobacillus, contributing to the deglycosylation and ring fission of flavonoids. CONCLUSION: SDPF promote SPFEs to transform to active metabolites probably by regulating gut microbiota. © 2023 Society of Chemical Industry.

p-Synephrine Indicates Internal Maturity of Citrus grandis (L.) Osbeck cv. Mato Peiyu-Reclaiming Functional Constituents from Nonedible Parts.

The processing of Citrus grandis Osbeck cv. Mato Peiyu (CGMP) fruits generates a considerable amount of waste, mainly the flavedo, albedo, and segment membrane; the generated waste yields severe environmental and economic challenges. In this study, we tried to reclaim some functional chemicals from the waste. Our data indicated that the essential oil content in the flavedo was 0.76-1.34%, with the major component being monoterpenes (93.75% in August, declining to 85.56% in November, including mainly limonene (87.08% to 81.12%) and others such as ß-myrcene). p-Synephrine (mg/100 g dry weight) declined accordingly (flavedo, 10.40 to 2.00; albedo, 1.80 to 0.25; segment membrane, 0.3 in August, 0.2 in September, and none since October). Polyphenols (in µg/g) included gallic acid (70.32-110.25, 99.27-252.89, and 105.78-187.36, respectively); protocatechuic acid (65.32-204.94, 26.35-72.35, and 214.98-302.65, respectively), p-coumaric acid (30.63-169.13, 4.32-17.00, and 6.68-34.32, respectively), ferulic acid (12.36-39.36, 1.21-10.25, and 17.07-39.63, respectively), and chlorogenic acid (59.19-199.36, 33.08-108.57, and 65.32-150.14, respectively). Flavonoids (in µg/g) included naringin (flavedo, 89.32-283.19), quercetin (181.05-248.51), nobiletin (259.75-563.7), hesperidin, and diosmin. The phytosterol content (mg/100 g) was 12.50-44.00 in the flavedo. The total dietary fiber in the segment membrane was 57 g/100 g. The antioxidant activity against the DPPH• and ABTS+• free radicals was moderately high. In conclusion, the waste of CGMP fruits is worth reclaiming for essential oil, p-synephrine, polyphenolics, and dietary fiber. Notably, p-synephrine content (flavedo: <8 mg/100 g dry weight, albedo: <2.0, or segment membrane: <0.4 mg) can serve as a marker of the internal maturation of CGMP fruits.

The aluminum distribution and translocation in two citrus species differing in aluminum tolerance.

BACKGROUND: Many citrus orchards of south China suffer from soil acidification, which induces aluminum (Al) toxicity. The Al-immobilization in vivo is crucial for Al detoxification. However, the distribution and translocation of excess Al in citrus species are not well understood. RESULTS: The seedlings of 'Xuegan' [Citrus sinensis (L.) Osbeck] and 'Shatianyou' [Citrus grandis (L.) Osbeck], that differ in Al tolerance, were hydroponically treated with a nutrient solution (Control) or supplemented by 1.0 mM Al3+ (Al toxicity) for 21 days after three months of pre-culture. The Al distribution at the tissue level of citrus species followed the order: lateral roots > primary roots > leaves > stems. The concentration of Al extracted from the cell wall (CW) of lateral roots was found to be about 8 to 10 times higher than in the lateral roots under Al toxicity, suggesting that the CW was the primary Al-binding site at the subcellular level. Furthermore, the Al distribution in CW components of the lateral roots showed that pectin had the highest affinity for binding Al. The relative expression level of genes directly relevant to Al transport indicated a dominant role of Cs6g03670.1 and Cg1g021320.1 in the Al distribution of two citrus species. Compared to C. grandis, C. sinensis had a significantly higher Al concentration on the CW of lateral roots, whereas remarkably lower Al levels in the leaves and stems. Furthermore, Al translocation revealed by the absorption kinetics of the CW demonstrated that C. sinensis had a higher Al retention and stronger Al affinity on the root CW than C. grandis. According to the FTIR (Fourier transform infrared spectroscopy) analysis, the Al distribution and translocation might be affected by a modification in the structure and components of the citrus lateral root CW. CONCLUSIONS: A higher Al-retention, mainly attributable to pectin of the root CW, and a lower Al translocation efficiency from roots to shoots contributed to a higher Al tolerance of C. sinensis than C. grandis. The aluminum distribution and translocation of two citrus species differing in aluminum tolerance were associated with the transcriptional regulation of genes related to Al transport and the structural modification of root CW.

Localization of CgVPE1 in secondary cell wall formation during tracheary element differentiation in the pericarp of Citrus grandis 'Tomentosa' fruits.

MAIN CONCLUSION: CgVPE1 is important in the differentiation of TE cells in C. grandis 'Tomentosa' fruits as it may directly affects secondary cell wall construction while participating in PCD. The vacuolar processing enzyme (VPE) plays an important role in both developmental and environmentally inducible programmed cell death (PCD); it was originally identified as a cysteine protease localized in the vacuole to activate and mature vacuolar proteins in plants. Interestingly, we found a VPE called CgVPE1 to be associated with deposition of the secondary cell wall in tracheary element (TE) cells in the pericarp of Citrus grandis 'Tomentosa' fruits. We then used ultrathin sections and the TUNEL assay to verify that PCD is involved in TE development. Furthermore, CgVPE1 was found to be mainly expressed in secretory cavities and TEs in the pericarp of Citrus grandis 'Tomentosa' fruits. Immunolocalization of CgVPE1 in the pericarp indicated that CgVPE1 is mainly distributed in the central large vacuole, endoplasmic reticulum, Golgi vesicles, cytosol, and secondary wall before TE maturation. CgVPE1 appeared earlier in the endoplasmic reticulum and Golgi vesicles of TEs cells. The vesicles containing CgVPE1 near the large central vacuole and secondary wall were observed, respectively. CgVPE1 proteins content in the cytoplasm decreased sharply, while the CgVPE1 content in the secondary cell wall did not change significantly after vacuole rupture. CgVPE1 protein contents in the secondary cell wall were significantly reduced until the TE cells developed into hollow thick-walled cells. Furthermore, labeling of VPE homologues in Arabidopsis thaliana using immunoelectron microscopy with anti-CgVPE1 antibody revealed that VPE homologues were specifically distributed in the secondary cell wall of stem TEs. Overall, these results suggested that CgVPE1 is not only involved PCD during TE cell development; furthermore, it may directly participate in the construction of plant secondary cell walls.

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.

Metabolomics combined with physiology and transcriptomics reveals how Citrus grandis leaves cope with copper-toxicity.

Limited data are available on metabolic responses of plants to copper (Cu)-toxicity. Firstly, we investigated Cu-toxic effects on metabolomics, the levels of free amino acids, NH4+-N, NO3--N, total nitrogen, total soluble proteins, total phenolics, lignin, reduced glutathione (GSH) and malondialdehyde, and the activities of nitrogen-assimilatory enzymes in 'Shatian' pummelo (Citrus grandis) leaves. Then, a conjoint analysis of metabolomics, physiology and transcriptomics was performed. Herein, 59 upregulated [30 primary metabolites (PMs) and 29 secondary metabolites (SMs)] and 52 downregulated (31 PMs and 21 SMs) metabolites were identified in Cu-toxic leaves. The toxicity of Cu to leaves was related to the Cu-induced accumulation of NH4+ and decrease of nitrogen assimilation. Metabolomics combined with physiology and transcriptomics revealed some adaptive responses of C. grandis leaves to Cu-toxicity, including (a) enhancing tryptophan metabolism and the levels of some amino acids and derivatives (tryptophan, phenylalanine, 5-hydroxy-l-tryptophan, 5-oxoproline and GSH); (b) increasing the accumulation of carbohydrates and alcohols and upregulating tricarboxylic acid cycle and the levels of some organic acids and derivatives (chlorogenic acid, quinic acid, d-tartaric acid and gallic acid o-hexoside); (c) reducing phospholipid (lysophosphatidylcholine and lysophosphatidylethanolamine) levels, increasing non-phosphate containing lipid [monoacylglycerol ester (acyl 18:2) isomer 1] levels, and inducing low-phosphate-responsive gene expression; and (d) triggering the biosynthesis of some chelators (total phenolics, lignin, l-trytamine, indole, eriodictyol C-hexoside, quercetin 5-O-malonylhexosyl-hexoside, N-caffeoyl agmatine, N'-p-coumaroyl agmatine, hydroxy-methoxycinnamate and protocatechuic acid o-glucoside) and vitamins and derivatives (nicotinic acid-hexoside, B1 and methyl nicotinate). Cu-induced upregulation of many antioxidants could not protect Cu-toxic leaves from oxidative damage. To conclude, our findings corroborated the hypothesis that extensive reprogramming of metabolites was carried out in Cu-toxic C. grandis leaves in order to cope with Cu-toxicity.

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck).

'Liuyuezaoyou' is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck 'Guanximiyou'. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

Adaptive Responses of Citrus grandis Leaves to Copper Toxicity Revealed by RNA-Seq and Physiology.

Copper (Cu)-toxic effects on Citrus grandis growth and Cu uptake, as well as gene expression and physiological parameters in leaves were investigated. Using RNA-Seq, 715 upregulated and 573 downregulated genes were identified in leaves of C. grandis seedlings exposed to Cu-toxicity (LCGSEC). Cu-toxicity altered the expression of 52 genes related to cell wall metabolism, thus impairing cell wall metabolism and lowering leaf growth. Cu-toxicity downregulated the expression of photosynthetic electron transport-related genes, thus reducing CO2 assimilation. Some genes involved in thermal energy dissipation, photorespiration, reactive oxygen species scavenging and cell redox homeostasis and some antioxidants (reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics) were upregulated in LCGSEC, but they could not protect LCGSEC from oxidative damage. Several adaptive responses might occur in LCGSEC. LCGSEC displayed both enhanced capacities to maintain homeostasis of Cu via reducing Cu uptake by leaves and preventing release of vacuolar Cu into the cytoplasm, and to improve internal detoxification of Cu by accumulating Cu chelators (lignin, reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics). The capacities to maintain both energy homeostasis and Ca homeostasis might be upregulated in LCGSEC. Cu-toxicity increased abscisates (auxins) level, thus stimulating stomatal closure and lowering water loss (enhancing water use efficiency and photosynthesis).

Microencapsulation Preservation of the Stability and Efficacy of Citrus Grandis Oil-Based Repellent Formulation against Aedes aegypti during Storage.

Essential oils have been widely used as an active ingredient in mosquito repellent products. However, essential oils are highly unstable and prone to degradation when exposed to the environment during storage. Microencapsulation techniques help to maintain the stability of molecules in essential oils that are sensitive to environmental stress, and therefore improve shelf life. In this study, the physical stability and efficacy of a repellent formulation consisting of encapsulated Citrus grandis essential oil (CGEO) were evaluated under different storage conditions over a 12-month period by comparing the formulation with a non-encapsulated formulation. The formulations were both stored under two different storage conditions, i.e., 25 ± 2 °C/60% ± 5% relative humidity (RH) and 40 ± 2 °C/75% RH ± 5%, for 12 months. Droplet size, zeta potential, and pH value were measured after 1, 6, and 12 months of storage to determine their stability. For the study of efficacy, each formulation was tested against Aedes aegypti under laboratory conditions. We found that the microencapsulated formulation's physical characteristics showed insignificant changes as compared with the non-encapsulated formulation during storage. The microencapsulated formulation demonstrated better repellent effects, sustaining high protection (>80%) for 4 more hours of exposure after 12 months of storage as compared with the non-encapsulated formulation that demonstrated high protection for only an hour post application. Microencapsulation helped to preserve the stability of the formulation, which resulted in high protection being maintained for over 12 months of storage.

[Herbalogical study on historical evolution of Juhong and Huajuhong].

In ancient times, there were two types of "Juhong" came from the tangerines(Citrus reticulata) and the pomelos(C. grandis and its cultivars), which corresponded to Juhong and Huajuhong recorded in the Chinese Pharmacopoeia respectively. In different periods, Juhong basically came from the same species and the same medicinal parts, but there were also some differences. This article sorted out the ancient and modern literature, under the guidance of "Succession theory of Medicinal materials varieties" and "Change theory of Medicinal materials varieties"(XIE Zong-wan), and combined with field investigation, the evolution and reasons of the original plants and medicinal parts of Juhong were analyzed. In the Han Dynasty and before, the peel of tangerines and pomelos were both used as medicine. In the Southern and Northern Dynasties, the way tangerine peel was used was dried and aged, and then "soaked in hot water and scraped off the mesocarp", which had the essence of only using exocarp as medicine of Juhong already, and its original plant was C. reticalata. In the Song Dynasty, the name of "Juhong" and its medicinal usage were recorded in book on materia medica, and the species and medicinal parts of tangerine were inherited from the previous dynasties. The way tangerine peel was used was only dried and aged without removing the mesocarp. The medicinal material obtained by the way was called Chenpi(dried and aged tangerine peel). The item "Juhong" listing as a separate medicinal material was first recorded in the Collected Discussions from Materia Medica(Bencao Huiyan) in the Ming Dynasty. In the Ming Dynasty, the Dao-di habitat of Juhong was recorded as Guangdong province in most books on materia medica, and the original plants probably were C. reticalata and C. grandis 'Tomentosa'(Huazhou pomelo, a special cultivated species of C. grandis produced in Huazhou, Guangdong, which was recorded in the Chinese Pharmacopoeia as "Huajuhong"), according to the records in the local chronicles. During the Qing Dynasty and the Republic of China, the original plants of Juhong were C. reticalata and C. grandis 'Tomentosa'. Of the two, the latter one was considered as the better. As far the medicinal part, it was still the exocarp, while the whole young fruit of C. grandis 'Tomentosa' began to be used as medicine. After the founding of The People's Republic of China, the exocarps of Citrus reticalata, C. grandis and C. grandis 'Tomentosa' were listed in the Chinese Pharmacopoeia under "Juhong". From the Northern and Southern Dynasties to the Republic of China, C. grandis exocarp was a fake of Juhong. Therefore, it was contradictory to historical records that C. grandis exocarp was listed in the Chinese Pharmacopoeia as Huajuhong. Juhong had been divided into two types as "Juhong" and "Huajuhong" since 1985. The medicinal part of Huajuhong was only the exocarp of immature and nearly mature fruits, but not the whole young fruit, the actual mainstream medicinal part of Huajuhong. The results are helpful to clarify the historical evolution of species and medicinal parts of Juhong and Huajuhong. It is suggested that in the next edition of Chinese Pharmacopoeia, only C. grandis 'Tomentosa' should be included as the original plant of Huajuhong, and C. grandis should be deleted, and the young fruit should be added in the medicinal parts besides the exocarp of immature and nearly mature fruit.

Valorization of pomelo (Citrus grandis Osbeck) peel: A review of current utilization, phytochemistry, bioactivities, and mechanisms of action.

Citrus grandis Osbeck, commonly known as "pomelo" or "shaddock," is the largest citrus fruit, the peel of which is a well-known agricultural residual waste. Pomelo peel offers a wide range of components such as essential oils, polysaccharides, and phytochemicals with potential food applications. Utilization of pomelo peel to recover these components is an important step toward agricultural sustainability. This review covers pomelo peel utilization opportunities beyond conventional composting and animal feed production, and critically examines value-added uses via the recovery of potentially bioactive components. The peel of pomelo accounts for approximately 30% of the total fruit weight and contains phytochemicals, including aroma-active volatiles, pectin, flavonoids, phenolic acids, carotenoids, coumarins, and polysaccharides. Recovery of these phytochemicals offers an opportunity for value-added utilization such as the development of enriched or functional foods and nutraceuticals. The health-promoting and therapeutic potential of pomelo peel extracts and isolated pure compounds have been evaluated through numerous in vitro and in vivo studies that revealed a wide range of bioactivities, including hypolipidemic, hypoglycemic, antioxidant, antimicrobial, anti-inflammatory, and anticancer effects. Preclinical evidence highlights multifaceted molecular and signaling events that possibly underlie the said bioactive potential. Overall, the pomelo processing industry offers a great opportunity to recover or produce valuable products from the large amounts of residual wastes it generates. It is envisaged that a thorough understanding of the bioactive components of pomelo peel, their functional and nutraceutical applications, and mode of actions will benefit the food industry.

[Identification and evaluation of Citrus grandis based on DNA barcode,UPLC and chromaticity method].

In order to identify the source of Citrus grandis and evaluate its quality originate from two areas comprehensively,DNA barcode was used to identify 26 samples of C. grandis. The content of naringin,rhoifolin,naringenin and apigenin was determined by UPLC method,and the color difference was numerically studied by color difference analyzer,which was related to the effective components of C. grandis. The results showed that samples was the source of C. grandis in both regions. The ITS2 sequence length was about400-500 bp,and the sequence similarity reached 99. 82%. There was only one base deletion in the two groups. There was one base A in some medicinal materials of Guangdong at 330 bp,but no base in Chongqing. The contents of naringin and rhoifolin in Chongqing samples were higher than those in Guangdong samples,and there were statistical differences between naringenin and apigenin. The chroma value showed that L*value of Guangdong was larger,a*value was smaller,L*value of Chongqing was smaller,and a*value was larger,while the b*value of both was not significantly different; The results of correlation analysis showed that naringin,rhoifolin,naringenin were positively correlated with L*,b*value,negatively correlated with a*value,and apigenin had no correlation with L*,a*,b*value. In this study,the scientific identification and evaluation of C. grandis was carried out to provide a new idea for the further study of the rapid identification and evaluation of C. grandis.

Low pH-responsive proteins revealed by a 2-DE based MS approach and related physiological responses in Citrus leaves.

BACKGROUND: Rare data are available on the molecular responses of higher plants to low pH. Seedlings of 'Sour pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were treated daily with nutrient solution at a pH of 2.5, 3, or 6 (control) for nine months. Thereafter, we first used 2-dimensional electrophoresis (2-DE) to investigate low pH-responsive proteins in Citrus leaves. Meanwhile, we examined low pH-effects on leaf gas exchange, carbohydrates, ascorbate, dehydroascorbate and malondialdehyde. The objectives were to understand the adaptive mechanisms of Citrus to low pH and to identify the possible candidate proteins for low pH-tolerance. RESULTS: Our results demonstrated that Citrus were tolerant to low pH, with a slightly higher low pH-tolerance in the C. sinensis than in the C. grandis. Using 2-DE, we identified more pH 2.5-responsive proteins than pH 3-responsive proteins in leaves. This paper discussed mainly on the pH 2.5-responsive proteins. pH 2.5 decreased the abundances of proteins involved in ribulose bisphosphate carboxylase/oxygenase activation, Calvin cycle, carbon fixation, chlorophyll biosynthesis and electron transport, hence lowering chlorophyll level, electron transport rate and photosynthesis. The higher oxidative damage in the pH 2.5-treated C. grandis leaves might be due to a combination of factors including higher production of reactive oxygen species, more proteins decreased in abundance involved in antioxidation and detoxification, and lower ascorbate level. Protein and amino acid metabolisms were less affected in the C. sinensis leaves than those in the C. grandis leaves when exposed to pH 2.5. The abundances of proteins related to jasmonic acid biosynthesis and signal transduction were increased and decreased in the pH 2.5-treated C. sinensis and C. grandis leaves, respectively. CONCLUSIONS: This is the first report on low pH-responsive proteins in higher plants. Thus, our results provide some novel information on low pH-toxicity and -tolerance in higher plants.

Molecular mechanisms underlying the participation of Ribonuclease T2 gene into self-incompatibility of Citrus grandis var. Shatianyu Hort.

The RIB (ribonuclease T2) of Citrus grandis var. Shatianyu Hort involved in self-incompatibility (SI) mechanism was identified by prokaryotic expression. RT-qPCR results showed that the expression level of RIB in self pollinated stigma is significantly higher than that in cross pollinated stigma. A vector for prokaryotic expression of RIB was constructed after codon-optimization, and the recombinant protein was induced and purified. In vitro pollen germination test indicated that the RIB protein markedly inhibited pollen germination and pollen tube growth. The result is helpful for better understanding of the molecular mechanism underlying the SI in C. grandis.

New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid from the pericarp of Citrus grandis (L.) Osbeck.

Two new 3-hydroxy-3-methylglutaryl (HMG) flavanone 7-O-diglycosides, cigranosides A and B (1 and 2), the known naringenin 7-(2''-α-rhamnosyl-6''-(3''''-hydroxy-3''''-methylglutaryl)-glucoside (melitidin, 3), their common biosynthetic precursor flavanone 7-O-diglycoside (naringin, 4), and one known flavone 7-O-diglycoside (rhoifolin, 5) were isolated from the pericarp of Citrus grandis (L.) Osbeck. The structures of these compounds were elucidated by spectroscopic and chemical techniques. The relative ratios and absolute configurations of the C-2 diastereomers of compounds 1, 2 and 4 were determined by online normal-phase HPLC-CD using a Chiralcel column. The absolute configuration of the HMG fragment in compounds 1-3 was assigned to be S through spectroscopic analysis of the mevalonamide obtained by amidation and reduction of the HMG moiety. The NO inhibitory activities of compounds 1-5 were evaluated using lipopolysaccharide-induced RAW264.7 cells. Compounds 1-5 were not cytotoxic to RAW264.7 cells at 10 µM.

Aluminum-responsive genes revealed by RNA-Seq and related physiological responses in leaves of two Citrus species with contrasting aluminum-tolerance.

Little is known about the physiological and molecular responses of leaves to aluminum (Al)-toxicity. Seedlings of Al-intolerant Citrus grandis and Al-tolerant Citrus sinensis were supplied daily with nutrient solution containing 0 mM (control) and 1.0 mM (Al-toxicity) AlCl3·6H2O for 18 weeks. We found that Al-treatment only decreased CO2 assimilation in C. grandis leaves, and that the Al-induced alterations of gene expression profiles were less in C. sinensis leaves than those in C. grandis leaves, indicating that C. sinensis seedlings were more tolerant to Al-toxicity than C. grandis ones. Al concentration was similar between Al-treated C. sinensis and C. grandis roots, but it was higher in Al-treated C. grandis stems and leaves than that in Al-treated C. sinensis stems and leaves. Al-treated C. sinensis seedlings accumulated relatively more Al in roots and transported relatively little Al to shoots. This might be responsible for the higher Al-tolerance of C. sinensis. Further analysis showed that the following several aspects might account for the higher Al-tolerance of C. sinensis, including: (a) Al-treated C. sinensis leaves had higher capacity to maintain the homeostasis of energy and phosphate, the stability of lipid composition and the integrity of cell wall than did Al-treated C. grandis leaves; (b) Al-triggered production of reactive oxygen species (ROS) and the other cytotoxic compounds was less in Al-treated C. sinensis leaves than that in Al-treated C. grandis leaves, because Al-toxicity decreased CO2 assimilation only in C. grandis leaves; accordingly, more upregulated genes involved in the detoxifications of ROS, aldehydes and methylglyoxal were identified in Al-treated C. grandis leaves; in addition, flavonoid concentration was increased only in Al-treated C. grandis leaves; (c) Al-treated C. sinensis leaves could keep a better balance between protein phosphorylation and dephosphorylation than did Al-treated C. grandis leaves; and (d) both the equilibrium of hormones and hormone-mediated signal transduction were greatly disrupted in Al-treated C. grandis leaves, but less altered in Al-treated C. sinensis leaves. Finally, we discussed the differences in Al-responsive genes between Citrus roots and leaves.

Proteome profile analysis of boron-induced alleviation of aluminum-toxicity in Citrus grandis roots.

Aluminum (Al)-toxicity and boron (B)-deficiency are two major factors limiting crop production in tropical and subtropical areas. Elevating B supply can alleviate the Al-induced inhibition of growth in Citrus grandis. Seedlings of C. grandis were irrigated for 18 weeks with nutrient solutions containing two B levels (2.5 and 20 µM H3BO3) and two Al levels (0 and 1.2 mM AlCl3·6H2O). By using 2-dimensional electrophoresis (2-DE) based MALDI-TOF/TOF-MS method, this study successfully identified and quantified sixty-one differentially abundant proteins in Citrus roots in response to B-Al interactions. The mechanisms underlying the B-induced alleviation of Al-toxicity unveiled by 2-DE technique could be summarized as follows: a) remodeling of cell wall by reducing the synthesis of lignin (sugar ATP Binding Cassette (ABC) transporter ATPase and cinnamyl alcohol dehydrogenase) and increasing the modification of cell wall (UDP-forming); b) enhancing the abundances of proteasomes and turnover of dysfunctional proteins (proteasome or protease); c) increasing the abundance of stress response proteins, such as alcohol dehydrogenase, S-adenosylmethionine synthetase (SAMS) and glycosyl hydrolase; d) reinforcing cellular biological regulation and signal transduction (calreticulin-1). For the first time, some proteins, such as cell division protein 48 (CDC48), calreticulin and phospholipase, which might be involved in the downstream signaling of Al in Citrus plants, were successfully identified.

Polysaccharides from Citrus grandis L. Osbeck suppress inflammation and relieve chronic pharyngitis.

Chronic pharyngitis, a common inflammation of the pharyngeal mucosa, is often caused by bacteria, viruses, alcohol abuse, overuse of the voice and cigarettes. This study aimed to explore the effects of polysaccharides of Citrus grandis L. Osbeck (PCG) in relieving chronic pharyngitis and illustrate the underlying mechanisms. Polysaccharides were obtained from PCG by column chromatographic extraction. Six clinical symptom scores, such as the severity of itchy throat, hoarseness, pain, odynophagia, cough and otalgia were evaluated in chronic pharyngitis patients after the oral intake of PCG. The effects of polysaccharides on chronic pharyngitis were investigated in ammonia-stimulated rabbits through pathology analysis. The levels of inflammatory markers in the peripheral blood T cells were analyzed by ELISA. The total and phosphorylated levels of ERK1/2, JNK and p38 were assessed by Western blot. Protein amount of IKKα and p65, IKKα/ß activity and p65 activity were evaluated by Western blot and luciferase assay. The clinical studies presented that PCG significantly relieved the six symptoms of chronic pharyngitis patients. Pathology analysis of chronic pharyngitis animals showed that the PCG treatment groups showed a significant decrease in the number of pathologic cells and the reduction of pathologic cells was dose-dependent (p < 0.01). ELISA analysis showed that PCG significantly inhibited the αCD3-induced increase of IFN-γ, IL-2 and IL-4 expression in a dose-dependent manner. Moreover, Western blot and luciferase assay suggested that the phosphorylation of IKKα/ß in peripheral blood T cells was inhibited by the administration of PCG. These results indicate that polysaccharides exhibit anti-inflammatory effects by inhibiting the phosphorylation of IKKs, subsequently suppressing the NF-κB pathway activation and decreasing the expression of inflammatory mediators.

Sulfur-Mediated-Alleviation of Aluminum-Toxicity in Citrus grandis Seedlings.

Limited data are available on the sulfur (S)-mediated-alleviation of aluminum (Al)-toxicity in higher plants. Citrus grandis seedlings were irrigated for 18 weeks with 0.5 mM MgSO4 or 0.5 mM MgSO4 + 0.5 mM Na2SO4, and 0 (-Al) or 1 mM AlCl3·6H2O (+Al, Al-toxicity). Under Al-toxicity, S decreased the level of Al in leaves; increased the relative water content (RWC) of roots and leaves, the contents of phosphorus (P), calcium (Ca) and magnesium (Mg) per plant, the dry weights (DW) of roots and shoots, the ratios of root DW/shoot DW, and the Al-induced secretion of citrate from root; and alleviated the Al-induced inhibition of photosynthesis via mitigating the Al-induced decrease of electron transport capacity resulting from the impaired photosynthetic electron transport chain. In addition to decreasing the Al-stimulated H2O2 production, the S-induced upregulation of both S metabolism-related enzymes and antioxidant enzymes also contributed to the S-mediated-alleviation of oxidative damage in Al-treated roots and leaves. Decreased transport of Al from roots to shoots and relatively little accumulation of Al in leaves, and increased leaf and root RWC and P, Ca, and Mg contents per plant might also play a role in the S-mediated-alleviation of Al-toxicity.