A newly isolated Trichoderma Parareesei N4-3 exhibiting a biocontrol potential for banana fusarium wilt by Hyperparasitism.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race4 (Foc TR4) is one of the most destructive soil-borne fungal diseases and currently threatens banana production around the world. Until now, there is lack of an effective method to control banana Fusarium wilt. Therefore, it is urgent to find an effective and eco-friendly strategy against the fungal disease. In this study, a strain of Trichoderma sp. N4-3 was isolated newly from the rhizosphere soil of banana plants. The isolate was identified as Trichoderma parareesei through analysis of TEF1 and RPB2 genes as well as morphological characterization. In vitro antagonistic assay demonstrated that strain N4-3 had a broad-spectrum antifungal activity against ten selected phytopathogenic fungi. Especially, it demonstrated a strong antifungal activity against Foc TR4. The results of the dual culture assay indicated that strain N4-3 could grow rapidly during the pre-growth period, occupy the growth space, and secrete a series of cell wall-degrading enzymes upon interaction with Foc TR4. These enzymes contributed to the mycelial and spore destruction of the pathogenic fungus by hyperparasitism. Additionally, the sequenced genome proved that strain N4-3 contained 21 genes encoding chitinase and 26 genes encoding ß-1,3-glucanase. The electron microscopy results showed that theses cell wall-degrading enzymes disrupted the mycelial, spore, and cell ultrastructure of Foc TR4. A pot experiment revealed that addition of strain N4-3 significantly reduced the amount of Foc TR4 in the rhizosphere soil of bananas at 60 days post inoculation. The disease index was decreased by 45.00% and the fresh weight was increased by 63.74% in comparison to the control. Hence, Trichoderma parareesei N4-3 will be a promising biological control agents for the management of plant fungal diseases.

Molecular physiology for the increase of soluble sugar accumulation in citrus fruits under drought stress.

To investigate the mechanism for drought promoting soluble sugar accumulation will be conducive to the enhancement of citrus fruit quality as well as stress tolerance. Fruit sucrose mainly derives from source leaves. Its accumulation in citrus fruit cell vacuole involves in two processes of unloading in the fruit segment membrane (SM) and translocating to the vacuole of fruit juice sacs (JS). Here, transcript levels of 47 sugar metabolism- and transport-related genes were compared in fruit SM or JS between drought and control treatments. Results indicated that transcript levels of cell wall invertase genes (CwINV2/6) and sucrose synthase genes (SUS2/6) in the SM were significantly increased by the drought. Moreover, transcript levels of SWEET genes (CsSWEET1/2/4/5/9) and monosaccharide transporter gene (CsPMT3) were significantly increased in SM under drought treatment. On the other hand, SUS1/3 and vacuolar invertase (VINV) transcript levels were significantly increased in JS by drought; CsPMT4, sucrose transporter gene 2 (CsSUT2), tonoplast monosaccharide transporter gene 2 (CsTMT2), sugar transport protein gene 1 (CsSTP1), two citrus type I V-PPase genes (CsVPP1, and CsVPP2) were also significantly increased in drought treated JS. Collectively, the imposition of drought stress resulted in more soluble sugar accumulation through enhancing sucrose download by enhancing sink strength- and transport ability-related genes, such as CwINV2/6, SUS2/6, CsSWEET1/2/4/5/9, and CsPMT3, in fruit SM, and soluble sugar storage ability by increasing transcript levels of genes, such as CsPMT4, VINV, CsSUT2, CsTMT2, CsSTP1, CsVPP1, and CsVPP2, in fruit JS.

Genome-wide analysis of citrus TCP transcription factors and their responses to abiotic stresses.

BACKGROUND: Citrus is one of the most important fruit crops in the world, and it is worthy to conduct more research on artificially controlling citrus plant growth and development to adapt to different cultivation patterns and environmental conditions. The plant-specific TEOSINTE BRANCHED1, CYCOLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are crucial regulators controlling plant growth and development, as well as responding to abiotic stresses. However, the information about citrus TCP transcription factors remains unclear. RESULTS: In this study, twenty putative TCP genes (CsTCPs) with the TCP domain were explored from Citrus sinensis genome, of which eleven (CsTCP3, - 4, - 5, - 6, - 10, - 11, - 15, - 16, - 18, - 19, - 20), five (CsTCP1, - 2, - 7, - 9, - 13), and four genes (CsTCP8, - 12, - 14, - 17) were unevenly distributed on chromosomes and divided into three subclades. Cis-acting element analysis indicated that most CsTCPs contained many phytohormone- and environment-responsive elements in promoter regions. All of CsTCPs were predominantly expressed in vegetative tissues or organs (stem, leaf, thorn, and bud) instead of reproductive tissues or organs (flower, fruit, and seed). Combined with collinearity analysis, CsTCP3, CsTCP9, and CsTCP13 may take part in leaf development; CsTCP12 and CsTCP14 may function in shoot branching, leaf development, or thorn development; CsTCP15 may participate in the development of stem, leaf, or thorn. In mature leaf, transcript levels of two CsTCPs (CsTCP19, - 20) were significantly increased while transcript levels of eight CsTCPs (CsTCP2, - 5, - 6, - 7, - 8, - 9, - 10, - 13) were significantly decreased by shading; except for two CsTCPs (CsTCP11, - 19), CsTCPs' transcript levels were significantly influenced by low temperature; moreover, transcript levels of two CsTCPs (CsTCP11, - 12) were significantly increased while five CsTCPs' (CsTCP14, - 16, - 18, - 19, - 20) transcript levels were significantly reduced by drought. CONCLUSIONS: This study provides significant clues for research on roles of CsTCPs in regulating citrus plant growth and development, as well as responding to abiotic stresses.

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.

Identification and function prediction of iron-deficiency-responsive microRNAs in citrus leaves.

Iron is a critical micronutrient for growth and development of plants and its deficiency limiting the crop productivity. MicroRNAs (miRNAs) play vital roles in adaptation of plants to various nutrient deficiencies. However, the role of miRNAs and their target genes related to Fe-deficiency is limited. In this study, we identified Fe-deficiency-responsive miRNAs from citrus. In Fe-deficiency conditions, about 50 and 31 miRNAs were up-regulated and down-regulated, respectively. The differently expressed miRNAs might play critical roles in contributing the Fe-deficiency tolerance in citrus plants. The miRNAs-mediated Fe-deficiency tolerance in citrus plants might related to the enhanced stress tolerance by decreased expression of miR172; regulation of S homeostasis by decreased expression of miR395; inhibition of plant growth by increased expression of miR319 and miR477; regulation of Cu homeostasis as well as activation of Cu/Zn superoxide dismutase activity due to decreased expression of miR398 and miR408 and regulation of lignin accumulation by decreased expression of miR397 and miR408. The identified miRNAs in present study laid a foundation to understand the Fe-deficiency adaptive mechanisms in citrus plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02669-z.

Reduced expression of CsPH8, a P-type ATPase gene, is the major factor leading to the low citrate accumulation in citrus leaves.

Citrate is an important intermediate product for the biosynthesis of several metabolites in plants. As two important organs of the citrus plant, fruits and leaves have their own metabolites characteristics; among them, citrate is normally high in fruit juice sacs (JS) and low in leaves. In this study, citrate content and transcript levels of citrate synthesis, transport, storage, and utilization related genes were compared between leaves and fruit JS of Citrus reticulata cv. 'Huagan No. 2', C. grandis cv. 'Hirado Buntan', and C. sinensis cv. 'Anliu'. Results indicated that the citrate content in fruit JS was significantly higher than in leaves of each cultivar. Only the relative mRNA levels of a P-type proton pump gene, CsPH8, was significantly lower in leaves than in fruit JS of three citrus cultivars, while other genes related to citrate biosynthesis, transport, storage, and utilization were highly expressed in leaves as compared to fruit JS. Furthermore, CsPH8 transient and stable transformation in leaves indicated that the change in citrate content is highly consistent with the change of CsPH8 transcript levels. Taken together, our results strongly suggest that the low accumulation of citrate in citrus leaves is mainly due to the low expression level of CsPH8; additionally, the high level of expression of citrate-utilizing genes would prevent citrate accumulation in the leaf organ.

Metabolite and Microbiome Profilings of Pickled Tea Elucidate the Role of Anaerobic Fermentation in Promoting High Levels of Gallic Acid Accumulation.

Gallic acid (GA) is an important active ingredient for its pharmacological activities. High levels of GA in tea can be obtained by anaerobic fermentation, but its mechanism is still unclear. Here, the profiles of metabolites and microbiomes in pickled tea were analyzed. The results showed that GA of pickled tea increased to 24.26 mg/g at 18 d after anaerobic fermentation, which was accompanied by the reducing levels of epicatechin gallate (ECG), epiafzelechin-3-O-gallate (EAG), and 7-galloylcatechin (7-GC) and the increasing relative abundances of Bacillus and other six bacterial genera. However, epigallocatechin gallate (EGCG) was basically stable during the whole fermentation process. These results suggested that EGCG contributes little to the GA formation during anaerobic fermentation, but ECG, EAG, and 7-GC should be the key precursors to form GA; moreover, bacteria, especially Bacillus, may be responsible for their bioconversion. It will establish an effective way to increase GA in tea production.

Multiomic Analysis Elucidates the Reasons Underlying the Differential Metabolite Accumulation in Citrus Mature Leaves and Fruit Juice Sacs.

Fruit and leaf possess distinctly different metabolites. Here, metabolites and transcriptome were compared between mature leaves (ML) and juice sacs (JS) of Citrus grandis "Hirado Buntan" to investigate the possible reasons. Results indicated that the remarkable difference in starch, total flavonoids and carotenoids, l-ascorbate, and jasmonic acid between ML and JS was tightly related to the expression levels of their biosynthesis-related genes, while the significant difference in abscisic acid and citrate was mainly related to the gene expression level(s) of 9-cis-epoxycarotenoid dioxygenase and proton pump genes, respectively. In addition, ATP citrate lyase probably plays a key role in the levels of flavonoids between ML and JS via providing different levels of acetyl-CoA. Taken together, these results identified some key candidate genes responsible for the content of a given metabolite and will contribute to research in regulating such metabolite content in citrus fruits.

Type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruit.

The aim of this work was to evaluate the general role of the vacuolar pyrophosphatase proton pump (V-PPase) in sucrose accumulation in citrus species. First, three citrus V-PPase genes, designated CsVPP-1, CsVPP-2, and CsVPP-4, were identified in the citrus genome. CsVPP-1 and CsVPP-2 belonging to citrus type I V-PPase genes are targeted to the tonoplast, and CsVPP-4 belonging to citrus type II V-PPase genes is located in the Golgi bodies. Moreover, there was a significantly positive correlation between transcript levels of type I V-PPase genes and sucrose, rather than hexose, content in fruits of seven citrus cultivars. Drought and abscisic acid treatments significantly induced the CsVPP-1 and CsVPP-2 transcript levels, as well as the sucrose content. The overexpression of type I V-PPase genes significantly increased PPase activity, decreased pyrophosphate contents, and increased sucrose contents, whereas V-PPase inhibition produced the opposite effect in both citrus fruits and leaves. Furthermore, altering the expression levels of type I V-PPase genes significantly influenced the transcript levels of sucrose transporter genes. Taken together, this study demonstrated that CsVPP-1 and CsVPP-2 play key roles in sucrose storage in the vacuole by regulating pyrophosphate homeostasis, ultimately the sucrose biosynthesis and transcript levels of sucrose transport genes, providing a novel lead for engineering or breeding modified taste in citrus and other fruits.

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.

Assessment of sugar and sugar accumulation-related gene expression profiles reveal new insight into the formation of low sugar accumulation trait in a sweet orange (Citrus sinensis) bud mutant.

The accumulation of soluble sugars in fleshy fruits largely determines their sweetness or taste. A spontaneous sweet orange mutant 'Hong Anliu' (HAL, Citrus sinensis) accumulates low soluble sugar content in fruit juice sacs than its wild type, 'Anliu' (AL) orange; however, the cause of reduced sugar content in 'HAL' fruit remains unclear. In this study, sugar content and expression profiles of genes involved in sugar metabolism and transport were compared between 'HAL' and 'AL' fruit juice sacs. In both cultivars, fructose and glucose displayed the increasing trends with significantly lower contents in 'HAL' than 'AL' after 160 DAF; moreover, sucrose had a declining trend in 'HAL' and increasing trend in 'AL' with fruit development. On the other hand, transcript levels of VINV, CWINV1, CWINV2, SUS4, SUS5, SPS1, SPS2, VPP-1, VPP-2, and some sugar transporter genes were significantly decreased in 'HAL' compared with 'AL' after 100 DAF or 160 DAF. Interestingly, the transcript levels of SPS2 and SUT2 exhibited a similar trend as it was found for sucrose content in both cultivars. These results suggested that the low sugar accumulation in 'HAL' fruit JS is accompanied by the reduced sink strength, sucrose-synthesis ability, and vacuolar storage ability compared with 'AL'; reduction of CWINVs, VINV, SPS2, SUT2, VPP-1, and VPP-2 transcript levels possibly plays a key role in the low storage of soluble sugars in the vacuoles of mutant juice sacs.

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.

CsPH8, a P-type proton pump gene, plays a key role in the diversity of citric acid accumulation in citrus fruits.

Citric acid homeostasis patterns and its content are diversified among the fruits of citrus cultivars, but the cause remained unclear. In this study we showed that changes of citric acid content were highly associated with the expression profiles of a P-type proton pump gene (CsPH8) in the fruits of six citrus cultivars; moreover, analysis of 21 different fruit samples indicated that the correlation coefficient between titratable acid content and CsPH8 transcript level was 0.5837 with a significant level (P < 0.05). Overexpression of CsPH8 in acidless pumelo juice sacs, strawberry fruit, and tomato fruit significantly increased the titratable acid or citric acid content besides the gene transcript level. On another hand, RNA interference of CsPH8 in acidic pumelo juice sacs significantly decreased the CsPH8 transcript level and the titratable acid or citric acid content as well. In addition, severe drought significantly increased the CsPH8 transcript level besides the titratable acid content. Taken together, these findings address the function of CsPH8 in citrus vacuolar acidification, confirm that CsPH8 plays a key role in the variation of citric acid content, and supported that the acid fluctuation influenced by drought, is at least partly due to the change of CsPH8 transcript level.

Genome-wide identification and transcript analysis of vacuolar-ATPase genes in citrus reveal their possible involvement in citrate accumulation.

The vacuolar H+-ATPase (V-ATPase) proton pump plays an important role in the acidification of vacuoles; however, genes encoding V-ATPase in the citrus genome and their roles in citric acid accumulation remain unclear in citrus fruit. In this study, we found at least one gene encoding subunit A, B, C, D, G, c'', d or e; two genes encoding the subunit E, F, H or a; and four genes encoding subunit c in the citrus genome. Spatial expression analysis showed that most genes were predominantly expressed in the mature leaves and/or flowers but were less expressed in root and juice cells. Two sweet orange (Citrus sinensis) cultivars, 'Anliu' (AL) and 'Hong Anliu' (HAL), which differ in terms of fruit acidity, were used in this study. The citric acid content was significantly higher in 'AL' fruits than in 'HAL' fruits over the entire experimental period (82 days-236 days after full blossom, DAFB). Transcript analysis showed that the transcript levels of most subunit genes, including V1-A, V1-B, V1-C, V1-E1, V1-G, V1-H2 and V0-a2, V0-c", V0-c4, and V0-d, were significantly lower in 'HAL' than in 'AL' fruits during fruit development and ripening. Moreover, ABA injection significantly increased the citric acid content, simultaneously accompanied by the obvious induction of V1-A, V1-C, V1-E1, V1-F1, V1-H2, V0-a1, V0-a2, V0-c1, V0-c2, V0-c4, and V0-d transcription levels. In conclusion, the results demonstrated that V1-A, V1-C, V1-E1, V1-H2, V0-a2, V0-c4, and V0-d may play more roles than other subunit genes in the vacuole acidification of citrus fruits. The lower activity of V-ATPase caused by the transcript reduction of some subunit genes may be one reason for the low citrate accumulation in 'HAL' juice sacs.

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.

Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant.

'Hong Anliu' (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of 'Anliu' (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between 'HAL' and 'AL'. Compared with 'AL', the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in 'HAL'. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H(+)-ATPase, vacuolar H(+)-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in 'HAL'. These results implied that 'HAL' has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with 'AL'. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in 'HAL'.

Citrus PH5-like H(+)-ATPase genes: identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits.

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.

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.