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‘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.

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.

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.

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.

Boron (B) deficiency inhibits and disturbs root growth and development by interacting with auxin (indole-3-acetic acid, IAA). However, the underlying mechanism of this interaction is still poorly understood. This study found that plants in a long-term boron deprivation treatment (∼0.25 µg L−1) had inhibited elongation of trifoliate orange roots, enlarged root tips and severe necrosis as well as a significant decrease in soluble boron and IAA content in seedling root tips. The results of a short-term boron deprivation treatment showed that a significant decrease in soluble boron and IAA content occurred after 3 hours of treatment (HOT) and 1 day of treatment (DOT), respectively. Moreover, the expression of IAA synthetic genes (TAA1, TAR2, YUC3 and YUC8) was strongly induced as early as 3 HOT and was then significantly reduced. The expression of rootward IAA transport genes (AUX1, PIN1 and PIN4) decreased significantly in the boron deprivation treatment, but the expression levels of shootward IAA transport genes (LAX1, ABCB1 and PIN3) were significantly increased. Taken together, the increase in IAA content before 1 DOT may be due to increased IAA synthesis caused by the induction of TAA1, TAR2, YUC3 and YUC8 expression; the subsequent decrease and the significantly lower final IAA content compared to the +B treatment (0.25 mg L−1 B) may be due to the reduced expression of IAA synthetic genes and rootward IAA transport genes and the increased expression of shootward IAA transport genes. Abbreviations: DAT: days after treatment; DOT: day of treatment; FW: fresh weight; HOT: hour of treatment; isB: insoluble boron; LC: liquid chromatography; LSD: least-significant difference; MS: mass spectrometry; PAT: polar auxin transport; qRT-PCR: quantitative real-time polymerase chain reaction; sB: soluble boron

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) 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.

The study aimed to determine if the variability of the effect of boron (B) deficiency on Newhall navel orange grafted on trifoliate orange (deficient-B-sensitive) and on citrange (deficient-B-tolerant) can be explained on the basis of changes in cell-wall content and in cell-wall B and calcium (Ca) concentrations. The plants were cultured in the nutrient solution (with or without B) for 65 days. Boron deficiency increased the cell-wall content in old leaves (leaves from last season) of trifoliate orange but had no impact on citrange. Boron deficiency did not reduce B concentration in cell walls of old leaves of citrange-grafted plants but increased their Ca concentration. For trifoliate-orange-grafted plants, however, B deficiency decreased the B concentration in cell walls of old leaves and did not increase their Ca concentration. The changes of B and Ca concentrations between with and without B supply in pectin were in good agreement with the changes of those in cell walls. The relatively greater abilit...

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.

Although citrus is an important source of ascorbic acid (AA), and even though girdling is an important agronomic practice in citrus production, the effect of girdling on ascorbate (ASC) accumulation has rarely been studied. In the present study, branch girdling (BG) was carried out on trees of Citrus unshiu cv. ‘Guoqing No. 1’ at 40 days before harvest to investigate its effect on fruit ASC accumulation and on the expression of genes involved in the AA l-galactose biosynthetic pathway. BG increased total ASC and AA contents in fruit peel and pulp. In parallel, soluble sugars in the fruit pulp increased. Moreover, the expression of all genes, except for l-galactose-1-P phosphatase (GPP), in the l-galactose pathway were induced in fruit pulp at least during the first 20 days of treatment while their expression (except for GPP) in fruit peel was reduced by BG treatment. Taken together, the expression profiles of six l-galactose biosynthetic genes did not coincide closely with the changes in fruit AS...

Citrus plants are frequently exposed to boron (B) deficiency, which has reduced citrus production worldwide. The effects of boron deficiency on citrus rootstock mineral absorption and allocation, and cell and vessel element anatomy are very important in understanding the nutrient absorbing mechanisms and resolving the boron deficiency problems of citrus production. Poncirus trifoliata seedlings were grown in Hoagland’s solution, which contained 0.25 µM H3BO3 (moderate B) and 0 µM H3BO3 (B deficiency). Seedling growth, root tip cell and vessel anatomy, and nutrient contents were investigated 30 days after treatment. B deficiency significantly inhibited the growth of Poncirus trifoliata seedlings, significantly decreased the mean lateral root length and root number, and significantly increased the root diameter and lateral root primordial (LRP) density. Ultrathin section micrographs showed a thickened root tip cell wall and more attachments on the cell wall in the boron-deficient treatment. The vessel wall of all root orders was markedly thickened and the inner vessel diameter decreased in the boron-deficient treatment. The boron and calcium contents in the leaf, stem and root were significantly decreased, and the phosphorus content decreased significantly in the leaf but not in stem or root in the boron-deficient treatment. The changes in root morphology and vessel element anatomical characteristics completely inhibited mineral nutrient absorption and transportation, resulting in a limited nutrient supply and reduced growth of Poncirus trifoliata in the boron-deficient treatment.

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.

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.

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.

Boron (B) toxicity reduces crop productivity and is a serious abiotic stress presenting in many parts of the world. MicroRNAs (miRNAs) play important roles in nutrient toxicity. In this study, we found that the B concentrations in roots and leaves of trifoliate orange (Poncirus trifoliata) were increased by 1.4- and 1.2-fold, respectively, after 10 days of excess B treatment (DAEBT). After 20 DAEBT, the B concentrations in roots and leaves increased by 2.8- and 2.0-fold, respectively. Transcript analysis showed that the miR397 relative transcript level decreased following the excess B treatment. Laccase7 (LAC7) was shown to be the target of miR397, and its transcription increased after the excess B treatment. In addition, the activity of laccase increased significantly following this treatment. Because LAC7 plays a role in lignin biosynthesis, we also measured the lignin concentrations in roots and leaves and found that they were increased following the excess B treatment. Our work demonstrates that decreased miR397 transcription plays a possible role in enhancing tolerance to B toxicity stress via negatively regulating LAC7 transcription and increasing the lignin concentration.

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.

Poncirus trifoliate is a traditional Chinese medicinal plant used for treating inflammation-related diseases for a long time and trifoliate orange contains abundant auraptene. The present study was to evaluate auraptene as a potential anti-inflammatory agent and investigate the mechanism of auraptene against prostaglandins E2 (PGE2) and cyclooxygenase-2 (COX-2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by comparing it with aspirin as a positive control group. The methods of enzyme-linked immunosorbent assay, reverse transcriptive polymerase chain reaction, real-time PCR, and western-blotting were used in the study. The results showed that auraptene exhibited better biocompatibility and lower cytotoxicity. At the same time, it significantly inhibited the production of PGE2 on LPS-stimulated macrophage cells. The auraptene-treated group had a higher COX-2 mRNA expression but relatively lower COX-2 protein level which implied that auraptene suppressed the post-transcriptional expression of COX-2 protein but not the transcriptional process. Compared with aspirin, the lower cytotoxicity of auraptene can make it a potential source for medicine that can benefit patients who are suffering from chronic inflammatory diseases and need long-term medication.

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.