A novel bud mutant of navel orange (Citrus sinensis) shows tolerance to chlorosis in acidic and magnesium-deficient soils.

Interveinal chlorosis in old leaves is a common occurrence in citrus orchards in southern China. The present study investigates the 'Langfeng' navel orange (LF, Citrus sinensis) grafted onto a Trifoliate orange (TO, Poncirus trifoliata) rootstock, which exhibits healthy green leaves, and the 'Newhall' navel orange (NHE, C. sinensis) grafted onto TO, which has typical magnesium (Mg) deficiency-induced chlorosis. Chemical analysis of the rhizosphere soil revealed that the pH values were around 3.92 and that both Mg and calcium (Ca) were significantly deficient in the rhizosphere soil of both grafting combinations (LF/TO and NHE/TO). Furthermore, the chlorotic leaves of NHE/TO had significantly lower levels of Mg, Ca, and phosphorus (P), and the green leaves of NHE/TO had significantly lower levels of Mg and Ca compared to the green leaves of the LF/TO. This suggests that Mg deficiency may be the primary cause of chlorosis in NHE/TO. A greenhouse study using the same graft combinations showed that the LF/TO plants had better growth than the NHE/TO, possibly by promoting Mg uptake and/or improving Mg distribution to leaves, thereby increasing carbon dioxide (CO2) assimilation and photosynthesis, optimizing carbohydrate distribution, and increasing plant biomass. This results in a phenotype that is tolerant to Mg deficiency. In conclusion, these findings suggest that the LF navel orange could be utilized in the development of new citrus varieties with improved Mg-use efficiency.

MYB308-mediated transcriptional activation of plasma membrane H + -ATPase 6 promotes iron uptake in citrus.

Iron-deficiency chlorosis is a common nutritional disorder in crops grown on alkaline or calcareous soils. Although the acclimation mechanism to iron deficiency has been investigated, the genetic regulation of iron acquisition is still unclear. Here, by comparing the iron uptake process between the iron-poor-soil-tolerant citrus species Zhique (ZQ) and the iron-poor-soil-sensitive citrus species trifoliate orange (TO), we discovered that enhanced root H + efflux is crucial for the tolerance to iron deficiency in ZQ. The H+ efflux is mainly regulated by a plasma membrane-localized H+-ATPase, HA6, the expression of which is upregulated in plants grown in soil with low iron content, and significantly higher in the roots of ZQ than TO. Overexpression of the HA6 gene in the Arabidopsis thaliana aha2 mutant, defective in iron uptake, recovered the wild-type phenotype. In parallel, overexpression of the HA6 gene in TO significantly increased iron content of plants. Moreover, an iron deficiency-induced transcription factor, MYB308, was revealed to bind the promoter and activate the expression of HA6 in ZQ in yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of MYB308 in ZQ roots significantly increased the expression level of the HA6 gene. However, MYB308 cannot bind or activate the HA6 promoter in TO due to the sequence variation of the corresponding MYB308 binding motif. Taking these results together, we propose that the MYB308 could activate HA6 to promote root H+ efflux and iron uptake, and that the distinctive MYB308-HA6 transcriptional module may be, at least in part, responsible for the iron deficiency tolerance in citrus.

Characteristics of boron distribution in the 'Newhall' navel orange plant with two root systems.

Grafting is a technique that provides a substantial way to enhance nutrient utilization thereby improves plant growth and yield quality. Although it is commonly practised in horticultural crops, the impact of scion-rootstock interaction on nutrient distribution is still unclear. Here, 'Newhall' navel orange plants grafted on Trifoliate orange (T) as the original rootstock were inarched with trifoliate orange (N/Tt combination) or carrizo citrange (N/Tc combination) rootstock seedlings. The experimental plants were treated with isotope 10B solution for 7 weeks to investigate the effect of different inarched rootstocks on B distribution and translocation by using a two-root system. From this study, the original rootstock played a more dominant role in B distribution to scion tissues than the inarching rootstock either in N/Tt or N/Tc combination. From inarched combinations, the carrizo citrange in the N/Tc combination had a higher ability to distribute B to new leaves, new twigs and old twigs than trifoliate orange in the N/Tt combination. However, the original rootstock of N/Tt distributed more B to scion tissues than N/Tc and the B concentration in old leaves and new leaves of N/Tt plants was significantly higher than that of N/Tc plants. These results suggest that scion B status is influenced by the B distribution of two inarching rootstocks in an inarching plant, as well as the affinity between the inarching rootstock and grafted plant. In addition, by either adding 10B to the inarching rootstock or original rootstock, we could detect 10B in the other rootstock root in both N/Tt and N/Tc combinations. The results further suggest that B can translocate from rootstock to leaves and then, re-translocate from scion to rootstock through the cycling of B transportation.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.

Foliar Supplied Boron Can Be Transported to Roots as a Boron-Sucrose Complex via Phloem in Citrus Trees.

Although foliar boron (B) fertilization is regarded as an efficient way to remedy B deficiency, the mechanisms of foliar B transport from leaves to roots are still unclear. In this study, performed with 1-year-old "Newhall" navel orange (Citrus sinensis) grafted on trifoliate orange (Poncirus trifoliata) plants, we analyzed the B concentration in leaves and roots, B-sucrose complex in the phloem sap after foliar application of 10B, girdling, and/or shading treatments. Results indicated that 10B concentration was significantly increased in roots after foliar 10B treatment. On the other hand, both girdling the scion stem and shading over the plants with a black plastic net significantly reduced the B and 10B concentration in roots. LC-MS analysis revealed that foliar 10B-treated plants had higher concentration of sucrose and some sugar alcohols in the phloem sap as compared to foliar water-treated plants. Combining with the analysis in the artificial mixture of B and sucrose, a higher peak intensity of the 10B-sucrose complex was found in the phloem sap of foliar 10B-treated plants compared to the control plants. Taken together, it is concluded that foliar B can be long distance transported from leaves to roots via phloem, at least by forming a B-sucrose complex in citrus plants.

Transcriptome Analysis Unravels Metabolic and Molecular Pathways Related to Fruit Sac Granulation in a Late-Ripening Navel Orange (Citrus sinensis Osbeck).

Lanelate navel orange (Citrus sinensis Osbeck) is a late-ripening citrus cultivar increasingly planted in China. The physiological disorder juice sac granulation often occurs in the fruit before harvest, but the physiological and molecular mechanisms underlying this disorder remain elusive. In this study, we found that fruit granulation of the late-ripening navel orange in the Three Gorges area is mainly caused by the low winter temperature in high altitude areas. Besides, dynamic changes of water content in the fruit after freezing were clarified. The granulation of fruit juice sacs resulted in increases in cell wall cellulose and decreases in soluble solid content, and the cells gradually became shrivelled and hollow. Meanwhile, the contents of pectin, cellulose, and lignin in juice sac increased with increasing degrees of fruit granulation. The activities of pectin methylesterase (PME) and the antioxidant enzymes peroxidase (POD), superoxide dismutase, and catalase increased, while those of polygalacturonase (PG) and cellulose (CL) decreased. Furthermore, a total of 903 differentially expressed genes were identified in the granulated fruit as compared with non-disordered fruit using RNA-sequencing, most of which were enriched in nine metabolic pathways, and qRT-PCR results suggested that the juice sac granulation is closely related to cell wall metabolism. In addition, the expression of PME involved in pectin decomposition was up-regulated, while that of PG was down-regulated. Phenylalanine ammonia lyase (PAL), cinnamol dehydrogenase (CAD), and POD related to lignin synthesis were up-regulated, while CL involved in cellulose decomposition was down-regulated. The expression patterns of these genes were in line with those observed in low-temperature treatment as revealed by qRT-PCR, further confirming that low winter temperature is associated with the fruit granulation of late-ripening citrus. Accordingly, low temperature would aggravate the granulation by affecting cell wall metabolism of late-ripening citrus fruit.

Identification of the magnesium transport (MGT) family in Poncirus trifoliata and functional characterization of PtrMGT5 in magnesium deficiency stress.

KEY MESSAGE: At least eight MGT genes were identified in citrus and PtrMGT5 plays important role in maintaining Mg homeostasis in citrus by getting involved in the Mg absorption and transport. Magnesium (Mg) is an essential macronutrient for plant growth and development, and the magnesium transporter (MGT) genes participate in mediate Mg2+ uptake, translocation and sequestration into cellular storage compartments. Although several MGT genes have been characterized in various plant species, a comprehensive analysis of the MGT gene family in citrus is still uncharacterized. In this study, eight PtrMGT genes were identified through genome-wide analyses. Phylogenetic analyses revealed that PtrMGT genes were classified into five distinct subfamilies. A quantitative RT-PCR analysis showed that eight PtrMGT genes were expressed in all of the detected tissues and they mainly expressed in the vegetative organs. Expression analyses revealed the PtrMGT genes responded to various Mg deficiency stresses, including absolute Mg deficiency and antagonistic Mg deficiency which caused by low pH or Al toxicity. PtrMGT5, which localizes to the plasma membrane and was transcriptionally active, was functionally characterized. PtrMGT5 overexpression considerably enhanced absolute Mg deficiency and antagonistic Mg deficiency tolerance in transgenic Arabidopsis plants, which was accompanied by increased fresh weight and Mg content, whereas opposite changes were observed when PtrMGT5 homolog in Valencia Orange callus was knocked down. Taken together, PtrMGT5 plays important role in maintaining Mg homeostasis in citrus by getting involved in the Mg absorption and transport.

Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus.

The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2-Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2-Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.

The Anti-Inflammatory Properties of Citrus wilsonii Tanaka Extract in LPS-Induced RAW 264.7 and Primary Mouse Bone Marrow-Derived Dendritic Cells.

'Zhique' (Citrus wilsonii Tanaka) is a traditional Chinese medicine. Its fruits have been used to treat inflammation-related symptoms, such as cough and sputum, though the underlying mechanism remains poorly understood. The aim of this study was to investigate the anti-inflammatory properties of 'Zhique' pulp extract (ZQE) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and primary mouse bone marrow-derived dendritic cells (BMDCs). The flavonoid profiles of the ZQE were determined by high performance liquid chromatography. The anti-inflammatory activity was evaluated in LPS-induced inflammatory RAW 264.7 macrophages and BMDCs through enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and Western blot assays. Naringin was a predominant flavonoid occurring in ZQE, followed by eriocitrin, hesperidin, neohesperidin, rhoifolin, naringenin, and poncirin. ZQE exhibited a very low cytotoxicity in LPS-stimulated RAW 264.7 macrophages. Meanwhile, ZQE significantly inhibited the production of prostaglandins E2 and secretion of cyclooxygenase-2 protein in LPS-stimulated RAW 264.7 macrophages, and markedly suppressed the mRNA expression of inflammatory mediators, such as cyclooxygenase-2, tumor necrosis factor alpha, interleukin-1 beta (IL-1ß), and IL-6 in LPS-induced RAW 264.7 macrophages and/or primary BMDCs. The ZQE inhibited the inflammatory responses in RAW 264.7 macrophages and BMDCs triggered by LPS. The results suggested that 'Zhique' has a high potential as a novel therapeutic agent to treat chronic inflammatory diseases.

Physiological and Transcriptional Changes of Three Citrus Rootstock Seedlings under Iron Deficiency.

Iron is an essential micronutrient for plants, and plants have evolved adaptive mechanisms to improve iron acquisition from soils. Grafting on iron deficiency-tolerant rootstock is an effective strategy to prevent iron deficiency-chlorosis in fruit-tree crops. To determine the mechanisms underlying iron uptake in iron deficiency, two iron deficiency-tolerant citrus rootstocks, Zhique (ZQ) and Xiangcheng (XC), as well as iron deficiency-sensitive rootstock trifoliate orange (TO) seedlings were studied. Plants were grown in hydroponics system for 100 days, having 50 µM iron (control) and 0 µM iron (iron deficiency) nutrient solution. Under iron deficiency, more obvious visual symptoms of iron chlorosis were observed in the leaves of TO, whereas slight symptoms were observed in ZQ and XC. This was further supported by the lower chlorophyll concentration in the leaves of TO than in leaves of ZQ and XC. Ferrous iron showed no differences among the three citrus rootstock roots, whereas ferrous iron was significantly higher in leaves of ZQ and XC than TO. The specific iron absorption rate and leaf iron proportion were significantly higher in ZQ and XC than in TO, suggesting the iron deficiency tolerance can be explained by increased iron uptake in roots of ZQ and XC, allowed by subsequent translocation to shoots. In transcriptome analysis, 29, 298, and 500 differentially expressed genes (DEGs) in response to iron deficiency were identified in ZQ, XC, and TO, respectively (Fold change ≥ 2 and Probability ≥ 0.8 were used as thresholds to identify DEGs). A Gene Ontology analysis suggested that several genotype-specific biological processes are involved in response to iron deficiency. Genes associated with cell wall biosynthesis, ethylene and abscisic acid signal transduction pathways were involved in iron deficiency responses in citrus rootstocks. The results of this study provide a basis for future analyses of the physiological and molecular mechanisms of the tolerance of different citrus rootstocks to iron deficiency.

Transcriptome Changes Associated with Boron Deficiency in Leaves of Two Citrus Scion-Rootstock Combinations.

Boron (B) deficiency stress is frequently observed in citrus orchards and causes considerable loss of productivity and fruit quality. Carrizo citrange (Cc) has been reported as a rootstock more tolerant to B deficiency than Trifoliate orange (To). The 'Newhall' navel orange (Ns) performed better when grafted onto Cc (Ns/Cc) than when grafted onto To (Ns/To) under long-term B deficiency. The present study confirmed that Ns/Cc had higher boron content, leaf fresh weight, lower leaf chlorosis and stronger photosynthesis ability than Ns/To. Moreover, B-deficiency significantly reduced the chlorophyll and carotenoid content in Ns/To. The content of total soluble sugar and lignin were dramatically increased and the expression levels of photosynthesis-related genes were substantially down-regulated in Ns/To by B-deficient treatment. B-deficiency also strongly induced expression levels of chlorophyll decomposition-related genes, glucose synthesis-related genes and lignin synthesis-related genes, and significantly inhibited the expression of carotenoid synthesis-related genes in Ns/To. Overall, these findings suggested that the influence of To on the scion of Ns was worse than that of Cc due to differently regulating these metabolic pathways under the long term of B-deficiency. The transcriptome analysis provided further information for understanding the mechanism of the different responses of scion-rootstock combinations to B-deficiency stress.

Identification and transcript profiles of citrus growth-regulating factor genes involved in the regulation of leaf and fruit development.

Growth-regulating factor (GRF) is an important protein in GA-mediated response, with key roles in plant growth and development. However, it is not known whether or how the GRF proteins in citrus to regulate organ size. In this study, nine citrus GRF genes (CsGRF1-9) were validated from the 'Anliu' sweet orange (AL, Citrus sinensis cv. Anliu) by PCR amplification. They all contain two conserved motifs (QLQ and WRC) and have 3-4 exons. The transcript levels of genes were detected by qRT-PCR. Transcript analysis showed that (1) CsGRF 1, 2, 5, 6, 7, and 9 expressed predominantly in young leaf, CsGRF 3 and 4 expressed predominantly in fruit immature juice sacs and CsGRF 8 expressed predominantly in root; (2) all citrus GRF genes had significantly higher expression in young leaves than mature leaf; (3) in juice sacs, the transcript levels of CsGRF1, 4, 5, 6, and 8 increased significantly while the transcript levels of CsGRF2, 3, 7, and 9 had no significant change from 80 DAF to 100 DAF. Besides, GA3 treatment did not affect the transcript levels of CsGRF5 and CsGRF6 but significantly increased the transcript levels of the other seven CsGRF genes in young leaves. These results suggested that all CsGRF genes involve in the leaf development, CsGRF1, 4, 5, 6, and 8 act developmentally whilst CsGRF2, 3, 7, and 9 play fundamental roles in fruit cell enlargement, which may be through GA pathway or GA-independent pathway.

Boron deficiency in woody plants: various responses and tolerance mechanisms.

Boron (B) is an essential microelement for higher plants, and its deficiency is widespread around the world and constrains the productivity of both agriculture and forestry. In the last two decades, numerous studies on model or herbaceous plants have contributed greatly to our understanding of the complex network of B-deficiency responses and mechanisms for tolerance. In woody plants, however, fewer studies have been conducted and they have not well been recently synthesized or related to the findings on model species on B transporters. Trees have a larger body size, longer lifespan and more B reserves than do herbaceous plants, indicating that woody species might undergo long-term or mild B deficiency more commonly and that regulation of B reserves helps trees cope with B deficiency. In addition, the highly heterozygous genetic background of tree species suggests that they may have more complex mechanisms of response and tolerance to B deficiency than do model plants. Boron-deficient trees usually exhibit two key visible symptoms: depression of growing points (root tip, bud, flower, and young leaf) and deformity of organs (root, shoot, leaf, and fruit). These symptoms may be ascribed to B functioning in the cell wall and membrane, and particularly to damage to vascular tissues and the suppression of both B and water transport. Boron deficiency also affects metabolic processes such as decreased leaf photosynthesis, and increased lignin and phenol content in trees. These negative effects will influence the quality and quantity of wood, fruit and other agricultural products. Boron efficiency probably originates from a combined effect of three processes: B uptake, B translocation and retranslocation, and B utilization. Root morphology and mycorrhiza can affect the B uptake efficiency of trees. During B translocation from the root to shoot, differences in B concentration between root cell sap and xylem exudate, as well as water use efficiency, may play key roles in tolerance to B deficiency. In addition, B retranslocation efficiency primarily depends on the extent of xylem-to-phloem transfer and the variety and amount of cis-diol moieties in the phloem. The B requirement for cell wall construction also contribute to the B use efficiency in trees. The present review will provide an update on the physiological and molecular responses and tolerance mechanisms to B deficiency in woody plants. Emphasis is placed on the roles of B reserves that are more important for tolerance to B deficiency in trees than in herbaceous plants and the possible physiological and molecular mechanisms of differential B efficiency in trees. We propose that B may be used to study the relationship between the cell wall and the membrane via the B-bridge. Transgenic B-efficient tree cultivars have considerable potential for forestry or fruit rootstock production on low B soils in the future.

Metabolic profiling reveals altered pattern of central metabolism in navel orange plants as a result of boron deficiency.

We focused on the changes of metabolite profiles in navel orange plants under long-term boron (B) deficiency using a gas chromatography-mass spectrometry (GC-MS) approach. Curling of the leaves and leaf chlorosis were observed only in the upper leaves (present before start of the treatment) of B-deficient plants, while the lower leaves (grown during treatment) did not show any visible symptoms. The metabolites with up-accumulation in B-deficient leaves were mainly proline, l-ornithine, lysine, glucoheptonic acid, fucose, fumarate, oxalate, quinate, myo-inositol and allo-inositol, while the metabolites with down-accumulation in B-deficient leaves were mainly serine, asparagine, saccharic acid, citrate, succinate, shikimate and phytol. The levels of glucose and fructose were increased only in the upper leaves by B deficiency, while starch content was increased in all the leaves and in roots. The increased levels of malate, ribitol, gluconic acid and glyceric acid occurred only in the lower leaves of B-deficient plants. The increased levels of phenols only in the upper leaves indicated that the effects of B on phenol metabolism in citrus plants may be a consequence of disruptions in leaf structure. Metabolites with opposite reactions in upper and lower leaves were mainly glutamine, glycine and pyrrole-2-carboxylic acid. To our knowledge, the phenomena of allo-inositol even higher than myo-inositol occurred characterized for the first time in this species. These results suggested that the altered pattern of central metabolism may be either specific or adaptive responses of navel orange plants to B deficiency.

Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLß1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLß1 encodes a putative ACL ß subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLß1 had 16 exons and 15 introns. CitACLα1 and CitACLß1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLß1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLß1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLß1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

Genome-wide identification and expression profile analysis of citrus sucrose synthase genes: investigation of possible roles in the regulation of sugar accumulation.

Sucrose synthase (Sus) (EC 2.4.1.13) is a key enzyme for the sugar accumulation that is critical to form fruit quality. In this study, extensive data-mining and PCR amplification confirmed that there are at least six Sus genes (CitSus1-6) in the citrus genome. Gene structure and phylogeny analysis showed an evolutionary consistency with other plant species. The six Sus genes contain 12-15 exons and 11-14 introns and were evenly distributed into the three plant Sus groups (CitSus1 and CitSus2 in the Sus I group, CitSus3 and CitSus6 in the Sus II group, and CitSus4 and CitSus5 in the Sus III group). Transcripts of these six CitSus genes were subsequently examined. For tissues and organs, CitSus1 and 2 were predominantly expressed in fruit juice sacs (JS) whereas CitSus3 and 4 were predominantly expressed in early leaves (immature leaves), and CitSus5 and 6 were predominantly expressed in fruit JS and in mature leaves. During fruit development, CitSus5 transcript increased significantly and CitSus6 transcript decreased significantly in fruit JS. In the fruit segment membrane (SM), the transcript levels of CitSus2 and 5 were markedly higher and the abundant levels of CitSus3 and 6 gradually decreased. Moreover, transcript levels of CitSus1-4 examined were higher and the CitSus5 transcript level was lower in the fruit SM than in fruit JS, while CitSus6 had a similar transcript level in fruit JS and SM. In addition, transcripts of CitSus1-6 responded differently to dehydration in mature leaves or to mild drought stress in fruit JS and SM. Finally, the possible roles of Sus genes in the regulation of sugar accumulation are discussed; however, further study is required.

Identification and transcript analysis of two glutamate decarboxylase genes, CsGAD1 and CsGAD2, reveal the strong relationship between CsGAD1 and citrate utilization in citrus fruit.

Glutamate decarboxylase (GAD, EC 4.1.1.15) has been suggested to be a key, regulatory point in the biosynthesis of γ-aminobutyrate (GABA) and in the utilization of citric acid through GABA shunt pathway. In this study we discovered two GAD genes, named as CsGAD1 and CsGAD2, in citrus genome database and then successfully cloned. Both CsGAD1 and CsGAD2 have a putative pyridoxal 5-phosphate binding domain in the middle region and a putative calmodulin-binding domain at the carboxyl terminus. Gene structure analysis showed that much difference exists in the size of exons and introns or in cis-regulatory elements in promoter region between the two GAD genes. Gene expression indicated that CsGAD1 transcript was predominantly expressed in flower and CsGAD2 transcript was predominantly expressed in fruit juice sacs; in the ripening fruit, CsGAD1 transcript level was at least 2-time higher than CsGAD2 transcript level. Moreover, CsGAD1 transcript level was increased significantly along with the increase of GAD activity and accompanied by a significant decrease of titratable acid (TA), suggesting that it is CsGAD1 rather than CsGAD2 plays a role in the citric acid utilization during fruit ripening. In addition, injection of abscisic acid and foliar spray of K2SO4 significantly increased the TA content of Satsuma mandarin, and significantly decreased GAD activity as well as CsGAD1 transcript, further suggesting the important role of CsGAD1 in the citrate utilization of citrus fruit.

A novel macromolecular extract screened from satsuma with pro-inflammatory effect.

Excessive consumption of horticultural fruit is a double-edged sword with both positive and negative effects. In Eastern countries, a large number of people have suffered from shang huo as a result of excessive consumption of "heating" foods, such as lychee, longan, mandarin orange, mango and civet durian. The present study adopted a step by step strategy screened the compositions with pro-inflammatory effect in satsuma fruits. The pro-inflammatory effects of all fractions were evaluated in RAW 264.7 cell lines by enzyme-linked immunosorbent assay (ELISA) and RT-PCR tests. The soluble water extract (SWE) from satsuma increased the production of prostaglandin E2 (PGE2) and promoted the expression level of cyclooxygenase-2 (COX-2) mRNA. SWE and high molecular weight molecules extracted from soluble water extract (HSWE) were respectively fractionated by dialysis bags and gel filtration chromatography. The macromolecular fraction named F1 was further obtained from HSWE, and could increase the production of inflammatory mediators. Finally F1 was resolved by SDS-PAGE and six proteins were identified by mass spectrometry. Compared with other detected proteins, polygalacturonase inhibitor (PGIP) and chitinase were the most likely candidate pro-inflammatory proteins according to molecular mass, and both of them were Citrus unshiu species. cDNA sequences of PGIP and chitinase were cloned and their functions were predicted as defensive proteins by SMART analysis. Excessive intake of these defensive proteins may result in adverse food reactions in human beings, such as shang huo and other immune responses.

Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.