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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.

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.

Inarching is often used to correct nutrient deficiency, but the characteristic of boron (B) uptake in inarched citrus remains unknown. One-year-old ‘Newhall’ navel orange plants on trifoliate orange were inarched with Carrizo citrange seedlings. After a 15-month B deficiency, the plants were resupplied with low B for 35 days to investigate the B uptake and the contribution of inarching rootstock to B absorption in different parts by using 10B labeling and split-root methods. When resupplying low B to B-deficient plants, the B concentration and proportion of newly absorbed B in new leaves, new twigs, and old leaves were higher in inarched than non-inarched plants. By contrast, the B concentration in original rootstock roots of inarched plants was lower than that of non-inarched plants. Regardless of B treatment, the fresh weight in original rootstock roots was significantly lower in inarched than non-inarched plants. Interestingly, the enriched 10B abundance was detected in the original rootstock from split-root treatments in which only the inarching rootstock was supplied with labeled 10B, implying that newly acquired B can be retranslocated from scion to rootstock. The newly-absorbed-B contribution of inarching rootstock was higher for the scion but lower for both rootstocks under low B conditions when compared with B-adequate conditions. These results suggest that, under low B conditions, inarching B-efficient Carrizo citrange onto B-deficient navel orange improves the plant's B uptake, increases the B concentration in young scion parts, and thus enhances the tolerance of the whole plant to B deficiency.

Chloride channels (CLCs) play pivotal roles in plant development and anion transport. However, little research has been conducted about the CLC in fruit-bearing plants. Here we provide an insight into the evolution and expression patterns of CLC gene family members in various tissues of trifoliate orange [Poncirus trifoliata (L.) Raf.] and their responses to several treatments. Genome-wide analysis identified six PtrCLC genes. The predicted proteins had similar numbers of amino acids, but shared a low sequence identity. Phylogenetic analysis revealed that PtrCLC were classified into two separate subgroups, and PtrCLC4 and PtrCLC6 in subgroup II were more closely related to bacterial CLCs. Sequence comparison with EcCLCA from Escherichia coli reveals that PtrCLC showed amino acid divergence in anion selectivity of CLC proteins. Real time qPCR analysis shows that PtrCLC genes, particularly PtrCLC6, preferentially expressed in leaves. Nitrogen deficiency irreversibly inhibited expression of PtrCLC genes except for PtrCLC1. In contrast, NaCl stress profoundly induced expression of PtrCLC genes, particularly PtrCLC2 and PtrCLC4, both of which were also upregulated by ABA treatment. The results presented here provide a solid foundation for a future functional research on citrus CLC genes.

Boron (B) is an essential microelement for vascular plants. Although it has frequently been reported that B deficiency leads to abnormal cell wall structure based on microscopic observation, what exactly occurs in the architecture of cell wall under this condition remains unknown. Navel orange plants that had been treated with different amounts of B were studied through chemical and instrumental (X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR)) analyses. Curling of the leaves and leaf chlorosis were observed only in the upper leaves of B-deficient plants. Boron deficiency significantly increased the relative hemicellulose and cellulose concentrations, and decreased covalently bound pectin in both upper and lower leaves. The results from XPS spectra suggested that the chemical states of carbon and oxygen were changed by B deficiency, and these changes were more serious in the upper leaves. The band at 3417 cm −1 in the upper leaf walls shifted to 3398 cm −1 due to B deficiency, suggesting that the mode of hydrogen bonding was changed by B deficiency (only in the upper leaves). The intensity and shape of the vibrations at 1200–900 cm −1 (the fingerprint region of polysaccharides) varied substantially between B-deficient plant cell walls and the control walls, indicating that B deficiency induced changes in both the amount and assembly of component polymers of cell wall. These results imply that the amount of wall components is not decisive for B deficiency symptoms in orange plants, but that rather structural changes within these fractions are important.

Boron efficiency of scion ‘Fengjie-72' is related to its less reduced boron concentration and distribution in leaves, achieved by decreasing the ratio of available boron in roots under boron-deficient conditions. Boron (B) deficiency is widespread in citrus orchards. Previous studies have demonstrated that the B-efficient navel orange scion ‘Fengjie-72' (Fs) and rootstock Carrizo citrange (Cr) are more tolerant to B deficiency than the closely related B-inefficient scion ‘Newhall’ (Ns) and rootstock trifoliate orange (Tr), respectively. However, the mechanisms underlying such differences remain unclear. Here, we investigated the differences in B distribution and forms among four combinations (Fs/Cr, Fs/Tr, Ns/Cr, and Ns/Tr) under adequate (0.25 mg/L) or deficient (0.001 mg/L) B supply for 300 days in sand culture. The results showed that B concentrations in buds and leaves of Fs-grafted plants were significantly higher than the respective concentrations of Ns-grafted plants under B-deficient conditions. Moreover, B distribution of Fs-grafted plants due to B deficiency was reduced less in leaves, but more in roots as compared to that of Ns-grafted plants. However, Ns/Cr accumulated more B in the scion stem (24 %) than the other combinations (17–19 %) when B was limited. A correlation was established between B efficiency and the ratio of B concentration in the rootstock stem or buds to the scion stem. Under B-deficient conditions, the ratio of available B (free B and semi-bound B) was significantly higher in leaves in Cr-grafted (36 %) than Tr-grafted plants (29 %), but lower in roots of Fs-grafted (22 %) than Ns-grafted plants (28 %). These results suggest that, under B-deficient conditions, differential B efficiency arises probably because Cr transports more B into scion, Fs redistributes B more efficiently within the plant, or both.

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.

Soil plastic film mulch is commonly employed in citrus production regions of East Asia to improve fruit quality. In the present study, Ponkan tangerine (Citrus reticulata Blanco) was mulched under the tree canopy with silver-black reflective film during fruit development. At about 12 days after mulching, total soluble sugar and citric acid contents in the segment membrane and/or juice sacs of fruit from mulched trees increased significantly relative to the control. In the segment membrane, the activities of acid invertase (AI) and sucrose synthase (SS; cleavage direction) increased significantly following mulch treatment. However, the activities of other enzymes, including neutral invertase, SS (synthetic direction) and sucrose phosphate synthase did not respond significantly under mulch treatment. In the juice sacs, SS activity (cleavage direction) from mulched treatments was significantly lower than that from control trees while SS activity (synthetic direction) from mulched trees was significantly higher than that from control trees. Moreover, the activities of cytoplasm aconitase (cyt-Aco) and isocitrate dehydrogenase (cyt-IDH) were significantly lower than those in the control fruits after 36 days of mulching. In conclusion, the activities of SS (synthetic direction) and AI were significantly enhanced while those of cyt-Aco and cyt-IDH were significantly reduced by soil plastic film mulch. A schematic model is present indicating the possible important roles that these key enzymes play in sugar and acid accumulation in citrus fruits under soil plastic film mulch.

SummaryBoron (B)-deficiency is a nutritional problem in the citrus industry worldwide. Under B-deficient conditions, symptoms such as corky split veins, small fruit, low tree vigour, and low fruit yield appear. In this study, we investigated the anatomical responses of mature leaves and the albedo of mature fruit of HB pummelo [Citrus grandis (L.) Osbeck] to B-deficiency. Under B-deficient conditions, the numbers of parenchyma cells in the vascular bundles of the leaf and fruit mesocarp increased significantly. The ratio of the area of the phloem to the area of the vascular bundle also increased, while the ratio of the area of the xylem to the area of the vascular bundle decreased in both tissues. Moreover, we observed alterations in the xylem elements under B-deficient conditions. The average lengths of xylem vessels were 216.7 µm and 175.4 µm in leaf main veins and in fruit mesocarp, respectively, both significantly lower than the corresponding values in B-sufficient control plants. However, B-deficienc...

Background and Aims In China, boron (B) deficiency is frequently observed in citrus orchards, and is responsible for considerable loss of productivity and quality. A better understanding of B distribution and remobilization within orange plants is important for developing programs in rational fertilization and effective mitigation of B-deficiency. In the present study (i) the distribution of newly absorbed B and (ii) the translocation of foliar-applied B in ‘Newhall’ navel orange grafted on citrange and trifoliate orange was investigated.

Citrus fruit is widely consumed and provides ascorbate for human health. The ascorbate content in pulp is generally higher in orange (Citrus sinensis Osb.) than in Satsuma mandarin (Citrus unshiu Marc.). However, what contributes to such difference is still unknown. In the present study, ascorbate accumulation, expression profiles of genes involved in L-galactose pathway and activity changes of enzymes related with L-ascorbic acid (AA) oxidation and recycling were investigated during fruit development and ripening in fruit pulp of Satsuma mandarin and orange. As fruit ripens, total ascorbate (T-ASC) or AA content increased in mandarin whereas fluctuated on a relatively high level in orange. Concentrations of T-ASC or AA in pulp of orange were over 1.5-fold higher than that in pulp of Satsuma mandarin during fruit ripening. Further analysis showed that each transcript of four genes (encoding GDP-D-mannose-3',5'-epimerase, GDP-L-galactose-pyrophosphatase, L-galactose dehydrogenase and L-galactono-1,4-lactone dehydrogenase respectively) in orange was almost on a higher level and the activities of oxidation enzymes (ascorbate oxidase and ascorbate peroxidase) were lower during fruit ripening as compared with Satsuma mandarin. As ascorbate pool size is decided by the combination of biosynthesis, oxidation and recycling, therefore, higher expression of four genes along with lower activity of oxidation enzymes should contribute at least partially to the higher ASC accumulation in orange pulp.

‘Yandangxueli’ is a pear cultivar with predominant citric acid in the ripe fruit, different from most of pear cultivars such as ‘Gengtouqing’ in which malic acid is the predominant acid type. It was found that ‘Yandangxueli’ accumulated citric acid for three times against that in ‘Gengtouqing’ at fruit ripening stage. To investigate the mechanism of citric acid accumulation in ‘Yandangxueli’, organic acids content, gene expression and enzyme activity were studied in both cultivars. Five genes, Pp:mtCs, Pp:cyAco, Pp:cyIdh, Pp:mtMdh and Pp:cyMe which encoded citric synthase (CS), cytosolic aconitase (cyACO), NADP-dependent isocitrate dehydrogenase (NADP-IDH), NAD-dependent malate dehydrogenase (NAD-MDH) and NADP-dependent malic enzyme (NADP-ME) respectively, were identified from pear fruit. Their expression profiles and the corresponding enzyme activities were determined throughout fruit development in both cultivars. Results from these enzymes indicated that there were no strict relationship between gene expression, enzyme activity and citric acid accumulation. Expression analysis for two Py:vVAtp genes encoding vacuolar H+-ATPase A subunit and one Py:vVpp gene encoding Vacuolar H+-pyrophosphatase showed that they were all with up-regulated expression at the later development stage of ‘Yandangxueli’ but with down-regulated expression in ‘Gengtouqing’. Therefore, it is concluded that the different ability in citric acid transportation and storage might be involved in the high citric acid content in ‘Yandangxueli’.

Boron (B) is an essential microelement for higher plants and has important physiological functions in plant growth and development. Citrus plants are frequently exposed to B-deficiency, but knowledge regarding the effects of B-deficiency on rootstock growth, root morphology and genotypic variations in citrus is limited. To evaluate the variations in plant-growth parameters in response to B-deficiency, five citrus rootstocks seedlings that grown in modified 1/2-strength Hoagland's solution that contained 0.25 μM H3BO3 (moderate B-level, Control) or 0 μM H3BO3 (B-deficiency, −B) were investigated before and 90 days after treatment. There are significant genotypic variations in plant-growth parameters (dry mass, leaf area and seedling height), root-morphological traits and B-concentrations. B-deficiency inhibited plant dry mass and leaf area expansion of Fragrant citrus, Sour orange and Trifoliate orange seedlings significantly (P 0.05). In addition, B-deficiency reduced the B-concentration in the leaves of all the five genotypes and the B-concentration in the roots of Fragrant citrus and Sour orange, none effect was observed on the B-concentration in stems of all studied genotypes and roots. However, there were significant relationships between the root-morphological traits and B-uptake efficiency. These results showed that Carrizo citrange and Red tangerine are B-efficient, Trifoliate orange is the moderate B-efficient genotypic rootstocks, whereas Fragrant citrus and Sour orange are B-inefficient genotypic rootstocks. In addition, Trifoliate orange was the moderate B-efficient rootstocks of five genotypes. The root-to-shoot dry mass ratio (R/S) and longer root numbers were higher in the Carrizo citrange and Red tangerine than that of Trifoliate orange, Fragrant citrus and Sour orange genotypes. That is to say, maintaining higher R/S and numerous longer lateral roots is very important in improving the B-efficiency and thus contribute much to the resistance of seedlings to B-deficiency in the Carrizo citrange and Red tangerine genotypes.

Information about citrus ethylene-responsive element binding factor (ERF) genes and their functions in fruit ripening or in stress tolerance is still scarce. In the present study, one of ERF genes, CitERF was isolated from fruit of Citrus unshiu with a maximal putative open reading frame encoding 207 amino acids. The deduced protein contains a region rich in acidic amino acids, an AP2/ERF domain and a KRRK nuclear localization signal. It belongs to group B of Class I in the ERF subfamily in which MdERF2 (Malus × domestica ethylene-response factor 2) and PsERF1b ( Prunus salicina ethylene-response factor 1b) were involved in the progress of fruit ripening. CitERF mRNA level in fruit peel and pulp increased obviously along with fruit ripening. However, its expression could be reduced significantly by treatments of total shading and fruit-bear-shoot girdling plus defoliation during fruit ripening. As for the response to abiotic stresses, CitERF expression was found to be induced continuously during the treatment of 10% polyethylene glycol. On the other hand, it could be induced to high level at 1 h after the treatment of 4 °C or 250 mM NaCl and then declined continuously. Taken together, the results suggested that CitERF may play an important role in some biological processes during fruit ripening and in improving tolerance to drought, low temperature and salt stress.

Field observations indicate that boron (B)-toxicity symptoms may occur in citrus plants from inappropriate foliar spraying or overfertilizing with B especially under low-rainfall conditions, where B can accumulate to levels that become toxic to plant growth. Previous work has indicated that different rootstocks can greatly influence the scion’s tolerance to B toxicity, however, little is known about the response of different citrus scion–rootstock combinations to excess-B conditions. In the present study, we investigated the effects of excess B on plant growth, gas exchange, B concentration, and distribution of four scion–rootstock combinations, Newhall and Skagg’s Bonanza navel orange (Citrus sinensis Osb.) scions grafted on Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.) and Trifoliate orange (Poncirus trifoliata L. Raf.) rootstocks. One-year-old plants of the four scion–rootstock combinations were grown for 183 d in sand–perlite (1 : 1, v/v) medium under greenhouse conditions. The plants were irrigated with half-strength Hoagland’s nutrient solution containing two B concentrations, 0.25 (control) and 2.50 (excess B) mg L–1. It was found that, apart from the combination of Newhall grafted on Carrizo citrange, the dry weights in various parts of the other three combinations were reduced by the excess-B treatment. Furthermore, the plants of Skagg’s Bonanza grafted on Carrizo citrange showed the highest growth reduction amongst the four scion–rootstock combinations. In most cases, the greater reductions in dry weight were found in roots as compared to the other plant parts under excess-B conditions, indicating that roots were more sensitive to B toxicity than the other tissues. In the case of Newhall plants grafted on Carrizo citrange, the entire plant growth was increased by excess-B treatment. Boron concentrations in all plants parts increased significantly by increasing the B supply in the nutrient solution. Leaves were the dominant sites of B accumulation and showed the greatest increase in B concentration compared to the other plant parts, as B concentration in the nutrient solution increased. Our results indicate that the combination of Newhall grafted on Carrizo citrange was more tolerant to B toxicity, while the combination of Skagg’s Bonanza grafted on Carrizo citrange was relatively more sensitive to B toxicity, in comparison with the other scion–rootstock combinations. However, Newhall plants contained more B in leaves and in roots than Skagg’s Bonanza plants when they were both grafted on Carrizo citrange, indicating that the mechanism underlying such great differential growth responses of the two scion–rootstock combinations to B toxicity may not be associated with B exclusion from roots or reduced translocation of B to shoots. Furthermore, B distribution in different plant parts implied that the mechanism was also unlikely related to altered distributions of accumulated B in plant tissues. However, inherent ability to tolerate excessive B concentration in plant tissues may be involved in B tolerance.

The effects of low boron (B) on plant growth, photosynthesis, B concentration and distribution of ‘Newhall’ orange ( Citrus sinensis Osb.) plants grafted on either Trifoliate orange ( Poncirus trifoliata (L.) Raf.) or Carrizo citrange [ C. sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] rootstocks were investigated. One-year-old plants of the two scion-rootstock combinations were grown for 183 days in sand:perlite (1:1, v/v) medium under greenhouse conditions. The plants were irrigated with half-strength Hoagland's nutrient solutions containing four B concentrations (0.01, 0.05, 0.10 and 0.25 mg l −1 ). The growth of root, stem of scion and leaves was less affected by low B treatments when ‘Newhall’ scion was grafted on Carrizo citrange than on Trifoliate orange. Thus, the growth of scions under low B conditions was mainly depended on the rootstock used, i.e., Carrizo citrange-grafted plants were more tolerant to low B compared to the plants grafted on Trifoliate orange. Boron concentrations in all plant parts decreased significantly by decreasing the B supply in the nutrient solution. Leaves were the dominant sites of B accumulation and showed the greatest reduction in B concentration compared to the other plant parts, as B concentration in the nutrient solution decreased. Irrespective of the rootstock, B levels in the upper–younger leaves were substantially higher than in basal-older leaves when plants were exposed to low B concentrations (≤0.05 mg l −1 ), suggesting that under such conditions B was preferentially translocated to upper–younger leaves to support their growth. Furthermore, B distribution in different plant parts indicated that there was a restriction in translocation of B from root to scion tissues (stems and leaves of scion) under conditions of limited B availability. In addition, low B treatments decreased leaf photosynthetic rate, stomatal conductance and transpiration rate but increased intercellular CO 2 concentration in the leaves of ‘Newhall’ plants, irrespective of the rootstock used.