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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.

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.

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.

It was reported that AtNIP5;1 is responsible for boron (B) uptake into the root under B-deficient conditions. In this study, we cloned an NIP5-like gene (named as CiNIP5 ) from Trifoliate orange. The full length of CiNIP5 was 1470 bp in which 903 bp formed the open reading frame, coding a protein of 300 amino acids. Sequence analysis showed that CiNIP5 had a highly conserved Asn-Pro-Ala (NPA) region and aromatic/arginine (ar/R) motif and it belongs to Nodulin 26-like intrinsic protein (NIP) group II. Analysis of gene expression indicated that CiNIP5 expressed mainly in the roots of citrus seedlings, and it increased obviously under B-deficiency but was suppressed under B toxicity. Additionally, the responses of CiNIP5 expression to B deficiency were compared between the roots of Carrizo citrange and Fragrant citrus, in which the former showed more tolerance than the latter to low B condition in field practice. CiNIP5 transcript level in the root of Carrizo citrange was induced and increased continuously to 7.7 times at 48 h after B deficient treatment (HABdT) as compared with that at 0 HABdT while in the root of Fragrant citrus it was induced to 4.4 times at 24 HABdT, and then decreased to half at 48 HABdT. The possible role of CiNIP5 was discussed in the mechanism of B-uptake efficiency of different rootstocks under B-deficiency.

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.

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.