Transcriptome Analysis Reveals Candidate Lignin-Related Genes and Transcription Factors during Fruit Development in Pomelo (Citrus maxima).

Juice sac granulation (a physiological disorder) leads to large postharvest losses of pomelo (Citrus maxima). Previous studies have shown that juice sac granulation is closely related to lignin accumulation, while the molecular mechanisms underlying this disorder remain elusive in pomelo. Our results showed that the lignin content in NC (near the core) and FC (far away from the core) juice sacs overall increased from 157 DPA (days post anthesis) to 212 DPA and reached a maximum at 212 DPA. Additionally, the lignin content of NC juice sacs was higher than that of FC juice sacs. In this study, we used transcriptome-based weighted gene co-expression network analysis (WGCNA) to address how lignin formation in NC and FC juice sacs is generated during the development of pomelo. After data assembly and bioinformatic analysis, we found a most correlated module (black module) to the lignin content, then we used the 11 DEGs in this module as hub genes for lignin biosynthesis. Among these DEGs, PAL (phenylalanine ammonia lyase), HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase), 4CL2 (4-coumarate: CoA ligase), C4H (cinnamate 4-hydroxylase), C3'H (p-coumarate 3-hydroxylase), and CCoAOMT1 (caffeoyl CoA 3-Omethyltransferase) were the most distinct DEGs in granulated juice sacs. Co-expression analysis revealed that the expression patterns of several transcription factors such as MYB, NAC, OFP6, and bHLH130 are highly correlated with lignin formation. In addition, the expression patterns of the DEGs related to lignin biosynthesis and transcription factors were validated by qRT-PCR, and the results were highly concordant with the RNA-seq results. These results would be beneficial for further studies on the molecular mechanism of lignin accumulation in pomelo juice sacs and would help with citrus breeding.

Identification and Functional Analysis of the CgNAC043 Gene Involved in Lignin Synthesis from Citrusgrandis "San Hong".

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of 'San hong' as the material, the staining for lignin with HCl-phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of 'San hong' by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

The complete chloroplast genome of Narcissus 'Pink Charm'.

The first complete chloroplast (cp) genome of Narcissus 'Pink Charm' was sequenced and characterized using Illumina paired-end data. The assembled cp genome was 159,988 in length with a GC content of 37.82%. A total of 137 genes were annotated, consisting of 91 protein-coding genes, eight ribosomal RNA genes, and 38 transfer RNA genes. The phylogenetic position based on the cp genome data revealed that Narcissus 'Pink Charm' is more closely related to Narcissus poeticus than other relative species.

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck).

'Liuyuezaoyou' is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck 'Guanximiyou'. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

Evaluation of phenolic composition and antioxidant properties of different varieties of Chinese citrus.

Citrus peels have health-promoting effects and are a rich source of antioxidant substances. This study evaluated the compositions of phenolic compounds and antioxidant activities in the peels of 52 citrus varieties with consistent planting time and management. The highest levels of total phenols (72.95 ± 37.60 mg/g DW) and total flavonoids (71.43 ± 37.64 mg/g DW) were found in mandarin. The highest phenolic acid content (18.78 ± 0.38 mg/g DW), dominated by protocatechuic acid, was found in kumquat. The antioxidant potency composite index was 6.23-94.56, suggesting mandarin varieties HJ, TWPG, TTPG, AY28, BZH and TCJC had the highest antioxidant activity. Statistics analysis indicated phenolic compounds and antioxidant activity were positively correlated. Principal component analysis and hierarchical cluster analysis suggested a strong relationship between phenolic compound composition and genetic background. This study indicated significant differences in the biological properties of various types of citrus peels; which are valuable for future utilization and research of citrus peels.

The complete chloroplast genome of 'Yulinglong' (Narcissus tazetta var. chinensis) with double-petaled in southeast of China.

In this study, the complete chloroplast (cp) genome of Narcissus tazetta var. chinensis cv 'Yulinglong' was sequenced and assembled by next-generation sequencing. The complete cp genome is 159,376 in length and contained 137 genes, consisting of 91 protein-coding genes, eight ribosomal RNA genes, and 38 transfer RNA genes. Phylogenetic analyses based on chloroplast genomes highly supported that 'Yulinglong' was evolutionarily close to Narcissus tazetta subsp.chinensis, which may provide more desirable information for the phylogenetic relationship between Narcissus tazetta var. chinensis and its relative species.

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses.

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis "Xuegan" plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

Reversing cold tumors to hot: An immunoadjuvant-functionalized metal-organic framework for multimodal imaging-guided synergistic photo-immunotherapy.

Immunotherapy assays using immunoadjuvants and tumor antigens could greatly increase the survival rates of patients with malignant tumors. As effective carriers, metal-organic frameworks (MOFs) have been widely utilized in cancer therapy due to their remarkable histocompatibility and low toxicity. Herein, we constructed a multimodal imaging-guided synergistic cancer photoimmunotherapy by employing a specific MOF (MIL101-NH2) as the core carrier; the MOF was dual-dressed with photoacoustic and fluorescent signal donors (indocyanine green, ICG) and immune adjuvants (cytosine-phosphate-guanine sequence, CpG) and named ICG-CpG@MOF. This nanocarrier could passively target the tumor site through the EPR effect and achieve multimodal imaging (fluorescence, photoacoustic, photothermal and magnetic resonance imaging) of the tumor. Synergistic cancer photoimmunotherapy was achieved via simultaneous photodynamic and photothermal methods with 808 nm laser irradiation. ICG-CpG@MOF achieved the GSH-controlled release of immunoadjuvant into the tumor microenvironment. Furthermore, the released tumor-associated antigen along with CpG could induce the transformation of tumor cells from cold to hot by activating the immune system, which significantly enhanced tumor cytotoxicity and achieved high cure rates with minimal side-effects. This strategy utilizing multimodal imaging and synergistic cancer photoimmunotherapy provides a promising approach for the diagnosis and treatment of cancer.

The complete chloroplast genome sequence of Citrus maxima (Burm.) Merr. 'Guanximiyou'.

Citrus maxima (Burm.) Merr. 'Guanximiyou' is a major citrus tree largely cultivated in China. A previous study has reported the complete chloroplast genome of C. maxima, but there may be some differences between wild species and cultivating variety. In this study, the complete chloroplast genome sequence of 'Guanximiyou' pummelo was characterized using BGISEQ-500 sequencing. The chloroplast genome was 160,186 bp in length and separated into four distinct regions such as large single-copy region (87,939 bp), small single-copy region (18,395 bp), and a pair of inverted repeat regions (26,926 bp). The genome contained a total of 109 genes including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Phylogenetic maximum-likelihood analysis revealed that 'Guanximiyou' pummelo was clustered with other Rutaceae species with high bootstrap values.

Risk factors for redetectable positivity in recovered COVID-19 children.

OBJECTIVE: To identify the risk factors for redetectable positivity (RP), and to provide a basis for prevention and control of coronavirus disease-2019 (COVID-19) in children. METHODS: A retrospective study was performed on all pediatric patients diagnosed with COVID-19. RP was defined as the positive result of real-time reverse transcriptase polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after symptom resolution and discharge. Children were defined as being less than 18 years old. RESULTS: Fourteen out of 38 (36.8%) pediatric patients exhibited RP. Compared with the non-RP group (n = 24), the RP group (n = 14) had more family cluster infections, relatively higher white blood cell (WBC) count and longer plasma prothrombin time (PT), while age and gender were insignificant. T lymphocyte subclassification was observed at five-time points: the first test after admission, 2 weeks, and 1, 2, and 3 months after discharge. The RP group had a higher percentage and count of CD8+ T lymphocytes and lower CD4+/CD8+ ratio at 2 weeks, while a lower percentage and count of CD4+ T lymphocytes and lower CD4+/CD8+ ratio at 2 months. The positive rate of nasopharyngeal swabs by RT-PCR was higher during the onset, while that of anal swabs was higher during the recovery of COVID-19. CONCLUSIONS: Family cluster infection, higher WBC count, and longer PT are the early risk factors for RP in recovered COVID-19 children. The dynamic changes in number and ratio of CD4+ and CD8+ T lymphocytes may be involved in prolonged SARS-CoV-2 clearance. Nasopharyngeal swabs sampling during the onset and anal swabs sampling during the recovery may improve the positivity rate of RT-PCR.

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri.

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

Characterization of the complete chloroplast genome of the Hongkong kumquat (Fortunella hindsii Swingle).

Hongkong qumquat (Fortunella hindsii Swingle) is a wild citrus species native to China. In this study, we firstly reporteded its complete chloroplast genome using BGISEQ-500 sequencing. The chloroplast genome is 160,145 bp in size, containing a large single copy region (87,467 bp), a small single copy region (18,730 bp), and a pair of IR regions (26,974 bp). The chloroplast genome contains 112 unique genes, including 79 protein-coding genes, 29 tRNAs, and 4 rRNAs. Phylogenetic maximum-likelihood analysis indicated that F. hindsii is closely related to Citrus species. The complete chloroplast genome would be subsequently used for citrus species researches.

Dissection of the Mechanism for Compatible and Incompatible Graft Combinations of Citrus grandis (L.) Osbeck ('Hongmian Miyou').

'Hongmian miyou' (Citrus grandis L. Osbeck) is mutated from 'Guanxi miyou', with a different spongy layer coloration. Trifoliate orange (Poncirus trifoliata) is widely used as rootstocks in 'Guanxi miyou' grafting, whereas 'Hongmian miyou' is incompatible with available trifoliate orange rootstocks. To explore the reasons for the etiolation of leaves of 'Hongmian miyou'/trifoliate orange, anatomical differences among different graft unions, gene expression profiles, and auxin levels of scion were investigated in this study. A histological assay indicated that there was no significant difference in anatomical structure between the compatible and incompatible combinations. A total of 1950 significant differentially-expressed genes (DEGs) were identified and analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction were significantly enriched. Moreover, the expression of nine genes in the auxin pathway were upregulated and three were downregulated in compatible combinations compared with those in the incompatible group. Further experiments verified that indole-3-acetic acid (IAA) content increases in the compatible graft combination, which suggests that IAA might promote graft compatibility.

Phylloremediation of Air Pollutants: Exploiting the Potential of Plant Leaves and Leaf-Associated Microbes.

Air pollution is air contaminated by anthropogenic or naturally occurring substances in high concentrations for a prolonged time, resulting in adverse effects on human comfort and health as well as on ecosystems. Major air pollutants include particulate matters (PMs), ground-level ozone (O3), sulfur dioxide (SO2), nitrogen dioxides (NO2), and volatile organic compounds (VOCs). During the last three decades, air has become increasingly polluted in countries like China and India due to rapid economic growth accompanied by increased energy consumption. Various policies, regulations, and technologies have been brought together for remediation of air pollution, but the air still remains polluted. In this review, we direct attention to bioremediation of air pollutants by exploiting the potentials of plant leaves and leaf-associated microbes. The aerial surfaces of plants, particularly leaves, are estimated to sum up to 4 × 108 km2 on the earth and are also home for up to 1026 bacterial cells. Plant leaves are able to adsorb or absorb air pollutants, and habituated microbes on leaf surface and in leaves (endophytes) are reported to be able to biodegrade or transform pollutants into less or nontoxic molecules, but their potentials for air remediation has been largely unexplored. With advances in omics technologies, molecular mechanisms underlying plant leaves and leaf associated microbes in reduction of air pollutants will be deeply examined, which will provide theoretical bases for developing leaf-based remediation technologies or phylloremediation for mitigating pollutants in the air.

Titanium as a Beneficial Element for Crop Production.

Titanium (Ti) is considered a beneficial element for plant growth. Ti applied via roots or leaves at low concentrations has been documented to improve crop performance through stimulating the activity of certain enzymes, enhancing chlorophyll content and photosynthesis, promoting nutrient uptake, strengthening stress tolerance, and improving crop yield and quality. Commercial fertilizers containing Ti, such as Tytanit and Mg-Titanit, have been used as biostimulants for improving crop production; however, mechanisms underlying the beneficial effects still remain unclear. In this article, we propose that the beneficial roles Ti plays in plants lie in its interaction with other nutrient elements primarily iron (Fe). Fe and Ti have synergistic and antagonistic relationships. When plants experience Fe deficiency, Ti helps induce the expression of genes related to Fe acquisition, thereby enhancing Fe uptake and utilization and subsequently improving plant growth. Plants may have proteins that either specifically or nonspecifically bind with Ti. When Ti concentration is high in plants, Ti competes with Fe for ligands or proteins. The competition could be severe, resulting in Ti phytotoxicity. As a result, the beneficial effects of Ti become more pronounced during the time when plants experience low or deficient Fe supply.

Differential Gene Expression in Rhododendron fortunei Roots Colonized by an Ericoid Mycorrhizal Fungus and Increased Nitrogen Absorption and Plant Growth.

Ericoid mycorrhizal (ERM) fungi are specifically symbiotic with plants in the family Ericaceae. Little is known thus far about their symbiotic establishment and subsequent nitrogen (N) uptake at the molecular level. The present study devised a system for establishing a symbiotic relationship between Rhododendron fortunei Lindl. and an ERM fungus (Oidiodendron maius var. maius strain Om19), quantified seedling growth and N uptake, and compared transcriptome profiling between colonized and uncolonized roots using RNA-Seq. The Om19 colonization induced 16,892 genes that were differentially expressed in plant roots, of which 14,364 were upregulated and 2,528 were downregulated. These genes included those homologous to ATP-binding cassette transporters, calcium/calmodulin-dependent kinases, and symbiosis receptor-like kinases. N metabolism was particularly active in Om19-colonized roots, and 51 genes were upregulated, such as nitrate transporters, nitrate reductase, nitrite reductase, ammonium transporters, glutamine synthetase, and glutamate synthase. Transcriptome analysis also identified a series of genes involving endocytosis, Fc-gamma R-mediated phagocytosis, glycerophospholipid metabolism, and Gonadotropin-releasing hormone (GnRH) signal pathway that have not been reported previously. Their roles in the symbiosis require further investigation. The Om19 colonization significantly increased N uptake and seedling growth. Total N content and dry weight of colonized seedlings were 36.6 and 46.6% greater than control seedlings. This is the first transcriptome analysis of a species from the family Ericaceae colonized by an ERM fungus. The findings from this study will shed light on the mechanisms underlying symbiotic relationships of ericaceous species with ERM fungi and the symbiosis-resultant N uptake and plant growth.

A New Oidiodendron maius Strain Isolated from Rhododendron fortunei and its Effects on Nitrogen Uptake and Plant Growth.

A new mycorrhizal fungal strain was isolated from hair roots of Rhododendron fortunei Lindl. grown in Huading Forest Park, Zhejiang Province, China. Morphological characterization and internal transcribed spacer rDNA analysis suggested that it belongs to Oidiodendron maius Barron, and we designated it as strain Om19. Methods for culturing Om19 were established, and the ability of Om19 to form mycorrhizae on R. fortunei was evaluated in a peat-based substrate. Microscopic observations showed hyaline hyphae on the surface of hair roots and crowded hyphal complexes (hyphal coils) inside root cortical cells of R. fortunei after inoculation, indicating that the roots were well colonized by Om19. In a second experiment, fresh and dry weight of R. fortunei 2 months after Om19 inoculation were greater than uninoculated plants, and the total nitrogen absorbed by plants inoculated with Om19 was greater than the uninoculated controls. qRT-PCR analysis of five genes related to N uptake and metabolism (two nitrate transporters, an ammonium transporter, glutamine synthetase, and glutamate synthase) showed that these genes were highly upregulated with twofold to ninefold greater expression in plants inoculated with Om19 compared to uninoculated plants. In the third experiment, Om19 was inoculated into the peat-based substrate for growing Formosa azalea (Rhododendron indica 'Formosa'). 'Formosa' azalea plants grown in the inoculated substrate had larger canopies and root systems compared to uninoculated plants. Our results show that Om19 could be an important microbial tool for improving production of Rhododendron plants.

Specific lignin accumulation in granulated juice sacs of Citrus maxima.

Juice sac granulation occurring in pummelo fruits [Citrus maxima (Burm.) Merr.] is an undesirable trait, and the underlying mechanism remains unresolved. Previous studies have shown that lignin metabolism is closely associated with the process of juice sac granulation. Here, a method suitable for lignin isolation from pummelo tissues is established. Acetylated lignins from different pummelo tissues and cultivars were analyzed by HSQC NMR. The results showed that lignins in granulated juice sacs were characterized by an extremely high abundance of guaiacyl units (91.13-96.82%), in contrast to lignins from other tissues, including leaves, stems, and segment membranes. The abnormally accumulated lignins in granulated juice sacs were specific and mainly polymerized from coniferyl alcohol. No significant difference was found in lignin types among various cultivars. These findings indicated that the mechanism of juice sac granulation might be similar among various cultivars, although very different degrees of juice sac granulation can be observed.

Epidemiology and clinical characteristics of hand foot, and mouth disease in a Shenzhen sentinel hospital from 2009 to 2011.

BACKGROUND: We investigated the epidemiological and clinical data of all hand, foot, and mouth disease (HFMD) cases in a sentinel hospital of Shenzhen, China from 2009 to 2011. METHODS: HFMD cases diagnosed in our institution were assessed from 2009 to 2011. Both epidemiological and clinical features were analyzed retrospectively. All the fatal cases were reported. RESULTS: A total of 12132 patients were diagnosed with HFMD, of which 2944 (24.3%) were hospitalized. Of the 2944 hospitalized patients, the highest proportion of diagnosed cases were admitted in May and July (989/2944, 33.6%). In 2009 all severe HFMD cases were diagnosed with enterovirus 71 (EV71). In 2010 and 2011, some of the severe HFMD were diagnosed with Coxsackievirus A16 (CA16). Incidence was highest in 0-4-year old children, with males being predominant. There were sporadic cases with HFMD the whole year except in February. All cases were cured in 2009. Six deaths were reported during 2010 and 2011. CONCLUSIONS: EV71 can cause severe complications and deaths in our region. HFMD is an important public health problem in Shenzhen in spite of stringent measures taken in preschool centers. A high degree of vigilance should be maintained over the disease situation.

Cloning of NAD-SDH cDNA from plum fruit and its expression and characterization.

A full-length cDNA consisting of 1444 bp for NAD dependent sorbitol dehydrogenase (NAD-SDH) was cloned from fruit of plum (Prunus salicina var. cordata cv. Younai) by means of RT-PCR and RACE. The cDNA containing an open reading frame (ORF) of 1101 bp encoded a polypeptide of 367 amino acid residues. The maltose binding protein fusion SDH (MBP-SDH) was expressed and partially purified from Escherichia coli cells, and biochemical properties of MBP-SDH and SDH cleaved from the fusion protein by factor Xa were characterized. The MBP-SDH had the specific affinity for NAD and was able to oxidize sorbitol, xylitol, l-ribitol and mannitol but not ethyl alcohol, arabitol and other polyols. The optimum pH for the oxidation of sorbitol and the reduction of fructose was 9.0 and 7.0, respectively; the maximum reaction rate occurred when temperature increased up to 50 °C in the presence of sorbitol. The MBP-SDH with a subunit of 80 kDa appears to be a hexamer. Its molecular weight was 478.6 kDa estimated by gel filtration and 493.2 kDa estimated using native linear gradient PAGE. The K(m) values for sorbitol, NAD, fructose and NADH were 95.86 mM, 0.31 mM, 1.04 M and 0.038 mM, respectively. However, when MBP was cleaved from the fusion enzyme, the SDH exists as a homotetramer with the native molecular weight of 164.8 kDa estimated by gel filtration. The K(m) values were 111.8 mM, 0.35 mM, 1.25 M and 0.048 mM for sorbitol, NAD, fructose and NADH, respectively. The MBP-SDH and the SDH were similar with respect to their kinetic characteristics despite their difference in quaternary structures.