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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.

PH5 is a petunia gene that encodes a plasma membrane H(+)-ATPase and determines the vacuolar pH. The citrate content of fruit cell vacuoles influences citrus organoleptic qualities. Although citrus could have PH5-like homologs that are involved in citrate accumulation, the details are still unknown. In this study, extensive data-mining with the PH5 sequence and PCR amplification confirmed that there are at least eight PH5-like genes (CsPH1-8) in the citrus genome. CsPHs have a molecular mass of approximately 100 kDa, and they have high similarity to PhPH5, AtAHA10 or AtAHA2 (from 64.6 to 80.9%). They contain 13-21 exons and 12-20 introns and were evenly distributed into four subgroups of the P3A-subfamily (CsPH1, CsPH2, and CsPH3 in Group I, CsPH4 and CsPH5 in Group II, CsPH6 in Group IV, and CsPH7 and CsPH8 in Group III together with PhPH5). A transcript analysis showed that CsPH1, 3, and 4 were predominantly expressed in mature leaves, whereas CsPH2 and 7 were predominantly expressed in roots, CsPH5 and 6 were predominantly expressed in flowers, and CsPH8 was predominantly expressed in fruit juice sacs (JS). Moreover, the CsPH transcript profiles differed between orange and pummelo, as well as between high-acid and low-acid cultivars. The low-acid orange \"Honganliu\" exhibits low transcript levels of CsPH3, CsPH4, CsPH5, and CsPH8, whereas the acid-free pummelo (AFP) has only a low transcript level of CsPH8. In addition, ABA injection increased the citrate content significantly, which was accompanied by the obvious induction of CsPH2, 6, 7, and 8 transcript levels. Taken together, we suggest that CsPH8 seems likely to regulate citrate accumulation in the citrus fruit vacuole.

Corky split vein caused by boron (B) deficiency in 'Newhall' Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1(st) phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2(nd) and 3(rd) phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.

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.

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.

The persistent consumption of citrus fruit mainly depends on its sweet–sour taste as well as pulp texture. To date, the simple facility by covering plastic film just over trees is commonly used in different citrus species for harvest time extension and quality improvement. In this study, the effect of covering trees of Citrus reticulata cv. Kinokuni with plastic films was investigated. Results indicated that simply covering plastic film over trees significantly decreased the soil water content, the light intensity, and then the photosynthetic rate, however, moderately increased the internal average temperature of 0–3 °C. Moreover, it significantly facilitated soluble sugar accumulation, increased water soluble pectin content, and decreased protopectin content. Investigating transcript profile showed that only 351 gene transcripts were significantly influenced by covering plastic film. Interestingly, most differently expressed genes (DEGs) were involved in carbohydrate and cell wall metabolisms except some DEGs were related to signal transduction and environmental adaption. Taken together, covering plastic film over trees comprehensively modified the internal environmental condition and improved the fruit sweetness and mastication trait by promoting soluble sugar accumulation, increasing water soluble pectin content, and decreasing protopectin content through the alteration of transcript level.

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 CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, 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, CitACLβ1, 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 CitACLβ1 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.

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 ( CsGRF 1–9) 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 CsGRF 1, 4, 5, 6, and 8 increased significantly while the transcript levels of CsGRF 2, 3, 7, and 9 had no significant change from 80 DAF to 100 DAF. Besides, GA3 treatment did not affect the transcript levels of CsGRF 5 and CsGRF 6 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, CsGRF 1, 4, 5, 6, and 8 act developmentally whilst CsGRF 2, 3, 7, and 9 play fundamental roles in fruit cell enlargement, which may be through GA pathway or GA-independent pathway.

The sucrose transporter (SUT) plays a major role in the transport of sucrose apoplastically. An extensive mining of the data confirmed that at least three SUT genes (CitSUT1, CitSUT2, and CitSUT3) were found in the databases of the three currently available citrus genomes. The exon and intron sizes varied among the three CitSUT genes, and the similarities among the genes were 40–50 %. The phylogenetic analysis revealed that the CitSUT1, CitSUT2, and CitSUT3 were distributed into groups SUT I, SUT II, and SUT III, respectively. The analysis of the spatiotemporal expression of the genes showed that the CitSUT1, CitSUT2, and CitSUT3 were predominantly expressed in the stamens, young leaves, and fruits [juice sacs (JS) and/or segment membrane (SM)], respectively. Moreover, in addition to the significant increase in sucrose levels from 114 days after anthesis (DAA) to 160 DAA, the transcript levels of CitSUT1 and CitSUT3 increased significantly in the JS and decreased significantly in the SM, whereas the CitSUT2 transcript levels decreased significantly either in the JS or in the SM. Additionally, treatment with ABA increased the accumulations of sucrose and fructose in fruits, which were accompanied by the induction of the CitSUT2 transcripts in fruits (JS and SM) and the CitSUT3 transcripts in the SM and the reduction of the CitSUT1 and CitSUT3 transcript levels in the JS. Thus, the CitSUT1 and CitSUT3 might play important roles in the transport of sucrose into the fruit JS during normal fruit development; the transcript alterations of the CitSUT2 and CitSUT3 under the ABA treatment might contribute to the increased accumulation of sucrose.

Iron (Fe) deficiency chlorosis is a yield-limiting problem in citrus production regions with calcareous soils. Physiological and transcriptional analyses of fragrant citrus ( Citrus junos Sieb. ex Tanaka) leaves from Fe-sufficient (IS) and Fe-deficient (ID) plants were investigated in this study. The physiological results showed that Fe, potassium, and nitrogen levels decreased by 12, 15, and 41% in ID leaves, respectively. However, zinc and copper levels increased by 49 and 35% in ID leaves, respectively. The chlorophyll (Chl) content, photosynthesis rate, stomatal conductance, and transpiration rate in ID leaves decreased by 55, 33, 38, and 42%, respectively, compared with IS leaves. Moreover, transcriptional profiling analysis showed that genes associated with Chl metabolism, photosynthesis, and nitrogen metabolism were dramatically downregulated by Fe deficiency. The expression of glutamyl-tRNA reductase 1, chlorophyll(ide) b reductase, and geranylgeranyl diphosphate reductase in ID leaves was 0.26–0.37 times that in IS leaves. The expression levels of 16 photosynthesis-related genes were severely downregulated by Fe deficiency. In addition, the transcription levels of nitrate transporter, nitrate reductase, and ferredoxin-nitrite reductase genes in ID leaves were 0.38–0.45 times those in IS leaves. Taken together, these results indicated that the block of Chl biosynthesis, the reduction of photosynthesis, and the repression of nitrogen absorption resulted in the chlorosis symptoms observed in fragrant citrus leaves.