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Huanglongbing (HLB), also known as citrus greening, is the most severe pandemics in citrus in more than 50 countries in Asia, Africa and America, causing serious economic losses worldwide (Gottwald, 2020; Wang, 2019). The disease is associated with a phloem-limited and fastidious member of the α-proteobacteriacea. 'Candidatus Liberibacter asiaticus' (CLas) is the most prevalent strain. It was frequently detected in leaves, stems and roots based on PCR detection which recognized both living and dead cells.

‘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.

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.

Iron (Fe) deficiency caused by calcareous soil is a serious problem in the cultivation of citrus (Citrus L.) trees. In this study, we report that ‘Zhique’ (Citrus wilsonii Tanaka) citrus rootstock from Chenggu county of Shaanxi province, China, shows tolerance to Fe deficiency under calcareous soil conditions. In the same calcareous field conditions, ‘Miyagawa Wase’ Satsuma mandarin (Citrus unshiu Marc.) grafted on trifoliate orange [Poncirus trifoliate (L.) Raf.] rootstock, the most commonly used rootstock, showed obvious interveinal chlorosis in young leaves, though some leaves or branches are asymptomatic, whereas no symptoms were found on those grafted on ‘Zhique’ rootstock. This was further evidenced by the fact that the chlorophyll concentration in chlorotic leaves of ‘Miyagawa Wase’ grafted on trifoliate orange was significantly lower than in those grafted on ‘Zhique’. In addition, transmission electron microscopy (TEM) analysis revealed a significant reduction of grana and stroma thylakoid of chloroplasts in chlorotic leaves. Measurement of Fe concentrations revealed that the total Fe and cell wall Fe showed no difference between ‘Zhique’ and trifoliate orange roots, whereas the ferrous Fe was significantly higher in ‘Zhique’ than trifoliate orange roots. Interestingly, both total Fe and ferrous Fe concentrations in chlorotic leaves were significantly lower than in green leaves of ‘Miyagawa Wase’ grafted on either ‘Zhique’ or trifoliate orange, whereas the cell wall Fe concentration of ‘Miyagawa Wase’ leaves only showed significant difference between the ‘Zhique’ and trifoliate orange samples. Further transcript assessment found that the Fe acquisition–related genes FIT, HA, FRO, and NRAMP were upregulated in roots of ‘Zhique’ compared with trifoliate orange, thus suggesting ‘Zhique’ might be more capable of Fe uptake under calcareous soil conditions. The novel citrus rootstock reported here could be used as an ideal material for Fe-uptake research, and as a Fe-deficiency-tolerant rootstock for citrus cultivation in calcareous soils.

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.

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

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 (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.

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...

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...

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.

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.

‘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.

Stone cell which appears in most pear cultivars contributes badly to pear internal quality. Lignin is the important component of stone cells and its first biosynthetic but specific step is catalyzed by cinnamoyl CoA reductase (CCR). In the present study, a cDNA encoding CCR, named as PpCCR (GenBank accession no. GU138672), was isolated from pear (Pyrus pyrifolia) fruit. Its full length is 1,247 bp with a putative maximal open reading frame of 1,017 bp, encoding a protein of 339 amino acids. Sequence alignment revealed that the deduced protein contained a typical CCR catalytic site and NADP(H) binding site. Phylogenetic analysis showed that PpCCR belonged to dicot CCR class II in which most CCRs participated in the regulation of lignin biosynthesis. PpCCR transcript could be detected in leaves, flowers and fruits. In addition, PpCCR expression profiles during fruit development were compared between two cultivars with significant difference in stone cell content in the flesh. Although PpCCR expressed in a similar pattern between the two cultivars and their transcript level consisted strictly with the change of lignin content, its transcript level was always higher in the high-lignin content cultivar than in the low-lignin content cultivar during fruit development. Therefore, it could be suggested that PpCCR is involved in the regulation of lignin biosynthesis and plays an important role in stone cell formation in pear flesh.

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.

Boron (B) is an essential microelement for higher plants, and its deficiency is widespread around the world and constrains the productivity of both agriculture and forestry. In the last two decades, numerous studies on model or herbaceous plants have contributed greatly to our understanding of the complex network of B-deficiency responses and mechanisms for tolerance. In woody plants, however, fewer studies have been conducted and they have not well been recently synthesized or related to the findings on model species on B transporters. Trees have a larger body size, longer lifespan and more B reserves than do herbaceous plants, indicating that woody species might undergo long-term or mild B deficiency more commonly and that regulation of B reserves helps trees cope with B deficiency. In addition, the highly heterozygous genetic background of tree species suggests that they may have more complex mechanisms of response and tolerance to B deficiency than do model plants. Boron-deficient trees usually exhibit two key visible symptoms: depression of growing points (root tip, bud, flower, and young leaf) and deformity of organs (root, shoot, leaf, and fruit). These symptoms may be ascribed to B functioning in the cell wall and membrane, and particularly to damage to vascular tissues and the suppression of both B and water transport. Boron deficiency also affects metabolic processes such as decreased leaf photosynthesis, and increased lignin and phenol content in trees. These negative effects will influence the quality and quantity of wood, fruit and other agricultural products. Boron efficiency probably originates from a combined effect of three processes: B uptake, B translocation and retranslocation, and B utilization. Root morphology and mycorrhiza can affect the B uptake efficiency of trees. During B translocation from the root to shoot, differences in B concentration between root cell sap and xylem exudate, as well as water use efficiency, may play key roles in tolerance to B deficiency. In addition, B retranslocation efficiency primarily depends on the extent of xylem-to-phloem transfer and the variety and amount of cis-diol moieties in the phloem. The B requirement for cell wall construction also contribute to the B use efficiency in trees. The present review will provide an update on the physiological and molecular responses and tolerance mechanisms to B deficiency in woody plants. Emphasis is placed on the roles of B reserves that are more important for tolerance to B deficiency in trees than in herbaceous plants and the possible physiological and molecular mechanisms of differential B efficiency in trees. We propose that B may be used to study the relationship between the cell wall and the membrane via the B-bridge. Transgenic B-efficient tree cultivars have considerable potential for forestry or fruit rootstock production on low B soils in the future.

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

Corky split vein can develop under long-term boron deficient conditions in Citrus sinensis L. Osbeck cv. Newhall. This symptom only occurs in the upper rather than the lower epidermis of old leaves. Our previous study demonstrated that vascular hypertrophy was involved in the symptoms, and the 3rd developmental stage of corky split vein (BD3) was the critical stage for phenotype formation. Here, we performed an intensive study on the BD3 vein and its control sample (CK3 vein). A lignin test demonstrated that the lignin content in BD3 vein was approximately 1.7 times more than the CK3 vein. Anatomical investigation of the corky split vein indicated that the upper epidermis was destroyed by overgrowing vascular cells, and the increased lignin may contribute to vascular cell differentiation and wounding-induced lignification. In a subsequent small RNA sequencing of the BD3 and CK3 veins, 99 known miRNAs and 22 novel miRNAs were identified. Comparative profiling of these miRNAs demonstrated that the 57 known miRNAs and all novel miRNAs exhibited significant expression differences between the two small RNAs libraries of the BD3 and CK3 veins. Associated with our corresponding digital gene expression data, we propose that the decreased expression of two miRNAs, csi-miR156b and csi-miR164, which leads to the up-regulation of their target genes, SPLs (csi-miR156b-targeted) and CUC2 (csi-miR164-targeted), may promote vascular cell division and orderless stage transition in old leaves.

ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, and/or CitACLα2 in cultivars ‘Niuher’ and ‘Owari’ increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in ‘Egan No. 1’ and ‘Iyokan’, although citrate content also decreased. In ‘HB pummelo’, in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLβ1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLβ1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

We focused on the changes of metabolite profiles in navel orange plants under long-term boron (B) deficiency using a gas chromatography-mass spectrometry (GC-MS) approach. Curling of the leaves and leaf chlorosis were observed only in the upper leaves (present before start of the treatment) of B-deficient plants, while the lower leaves (grown during treatment) did not show any visible symptoms. The metabolites with up-accumulation in B-deficient leaves were mainly proline, l-ornithine, lysine, glucoheptonic acid, fucose, fumarate, oxalate, quinate, myo-inositol and allo-inositol, while the metabolites with down-accumulation in B-deficient leaves were mainly serine, asparagine, saccharic acid, citrate, succinate, shikimate and phytol. The levels of glucose and fructose were increased only in the upper leaves by B deficiency, while starch content was increased in all the leaves and in roots. The increased levels of malate, ribitol, gluconic acid and glyceric acid occurred only in the lower leaves of B-deficient plants. The increased levels of phenols only in the upper leaves indicated that the effects of B on phenol metabolism in citrus plants may be a consequence of disruptions in leaf structure. Metabolites with opposite reactions in upper and lower leaves were mainly glutamine, glycine and pyrrole-2-carboxylic acid. To our knowledge, the phenomena of allo-inositol even higher than myo-inositol occurred characterized for the first time in this species. These results suggested that the altered pattern of central metabolism may be either specific or adaptive responses of navel orange plants to B deficiency.

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 investigate the possible involvement of calcium in the process of flower differentiation, calcium ion in the cells of apical meristem was detected electronmicroscopically using buds on spurs and vegetative shoots of Japanese pear (Pyrus pyrifolia Nakai) cv. Shinsui as materials.1. Cells at samples fixed with glutaraldehyde-formaldehyde and post-fixed with osmium tetroxide, both of which contained pyroantimonate, precipitated many black particles whereas they were not observable in photomicrographs of cells fixed with fixatives without antimonate. Most of the black particles disappeared when the preparations were treated with EGTA which indicate that the black particles are calcium-antimonate complexes. The localization, density and the size of the particles within cells of apical meristem changed dramatically as flower differentiation proceeded.2. Density of calcium particles differed depending on the activity of the cells. Until June 5, only a few particles were observed; they were mainly localized in plastids of cells from buds on spurs. The particles subsequently increased, and then decreased temporarily just before the onset of floral differentiation; another increase to a maximum level on June 26 was noted at which time corpus cells became very active judging from dense staining of cytoplasm and increased dictiosomes and endoplasmic reticulum (ER). The particles decreased thereafter through 3 and 10 July and reached a low value on July 17 when calyx primordia began to differentiate.3. Ca2+ density in nuclei of cells was low before flower differentiation, but it increased, indicative that floral differentiation was occurring. Within nucleus, Ca2+ distri- buted mainly in nuclear fluid, some in granular region of nucleolus and its releasing materials, but not in the fibrous area of nucleolus, chromosomes or chromatin.4. Although Ca2+ is mainly concentrated in the cell wall, its level in the cell wall decreased as Ca2+ in the vacuoles increased at the floral differentiation stage. The vacuole normally contains little Ca2+, but during this period it increased dramatically.5. Plastids contained almost all Ca2+ in the cell before June 5, and after that, continued to be one of the main distribution area of Ca2+. But Ca2+ in plastids decreased when Ca2+ in cells markedly increased on June 26. On the other hand, Ca2+ contents in mitochondria, dictiosomes, and ER changed parallel to the changes of Ca2+ in cytoplasm, and increased after flower differentiation stage on June 26.6. The size of the Ca2+ particles also changed during flower differentiation. The size became smaller as the flower differentiation proceeded, smallest on July 10, but after that became larger when Ca2+ density rapidly decreased.7. Possible role of Ca2 + in the cell of apical meristem in flower differentiation of Japanese pear was discussed.

To elucidate the mechanism of flower differentiation, ultrastructural changes, particularly the changes in amount and shape of organelles were investigated in the apical meristem of the buds of Japanese pear (Pyrus pyrifolia Nakai cv. Shinsui) during flower bud differentiation.1. The most important stages in the flower bud differentiation were from the beginning of activation of corpus cells on June 26 to the differentiation of sepal primordia on July 17. During these periods all the organelles in the cell underwent remarkable changes.2. During flower differentiation the number of dictiosomes markedly increased with concomitant cytomorphological changes such as many larger vesicles and the reduction in length of dictiosome itself. Distribution of endoplasmic reticulum (ER) also increased with the increase in ribosome. ER evolved into a kind of network from a discontinuous linear shape. There also existed active dictiosomes associated with ER.3. The number and size of mitochondria increased during flower differentiation. As the size increased the shape became spherical or oval. The number and size of plastids in the cells of active tissues did not differ between flower buds and vegetative buds. The size of plastids in the cells of quiescent zone changed markedly, increasing initially and then decreasing, because the size was closely related to the amount of starch grains the plastids contained.4. While flower differentiation progressed, chromatin in the nucleus increased and became densely stained in the cells of apical meristem of the flower buds. Nucleoli also increased in size, and occasionally two nucleoli were observed in a nucleus. Actively dividing cells in the apical meristem of flower buds were also characterized by many small vacuoles.5. These ultrastructural changes of the organelles in the cell of flower buds were discussed in relation to their physiological roles.

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.

Excised shoot-tips produced from banana plants belonging to cv. Guangdong No.1 (ABB group) were cryopreserved successfully by vitrification using the PVS2 solution. Ultrastructural of banana shoot-tips cells was also observed by using electron micryoscopy (TEM). The results showed that the plasmolysis became more and more severe during the course of dehydration. Cells were mainly damaged during the freezing and thawing process. Most cell protoplasts condensed, and cell organelles, cell membranes and nucleus envelopes were lethally injured after cryopreservation. But only a few cells located in the meristematic dome arose reversible process although their structures were varied. They could survive and regenerate plantlets after freezing conservation.