Genome-wide identification and characterization of DEAD-box helicase family associated with early somatic embryogenesis in Dimocarpus longan Lour.

DEAD-box (DDX) proteins belong to the largest subfamily of RNA helicase SF2, which contributes to all biological processes of RNA metabolism in the plant kingdom. Till now, no significant data are available regarding studies on DDX in Somatic Embryogenesis (SE) of woody plants. It is important to investigate the biological function of the DlDDX family in longan SE. Thus, a comprehensive analysis of 58 longan DEAD-box (DlDDX) genes characterization was performed by genome-wide identification and transcript abundance validation analysis. Homologous evolution has revealed that some DlDDXs in longan had high sequence similarity with Mus musculus, Citrus and Saccharomyces cerevisiae, indicating that DlDDXs were highly conservative in the animal, plant, and microorganism. Remarkably, gene duplication, purifying selection, and alternative splicing events, and new auxiliary domains have likely contributed to the functional evolution of DlDDX, indicating that DlDDX appeared neofunctionalization in longan. Besides, DlDDX3, 15, 28, 36 might interact with protein complex (MAC3A, MAC3B, CDC5, CBP20) of miRNA biosynthesis. Notably, DlDDX28 contained a novel auxiliary domain (CAF-1 p150), which might contribute to DNA demethylation in longan early SE. 4 DlDDX genes significantly expressed not only in early SE and zygotic embryogenesis (ZE) but also up-regulated at high levels in 'Honghezi' and 'Quanlongbaihe' with abortive seeds, which are of great significance. Moreover, some DlDDXs presented abiotic stress-response dynamic expression patterns by ABA, SA, JA, and NaCl treatments during early SE. Hence, DEAD-box is essential to SE development and seed abortive in longan.

Soluble Acid Invertases Act as Key Factors Influencing the Sucrose/Hexose Ratio and Sugar Receding in Longan ( Dimocarpus longan Lour.) Pulp.

Soluble acid invertases (SAIs) cleave sucrose into hexose in vacuoles and play important roles in influencing fruit quality. However, their potential roles in regulating sugar composition and the "sugar receding" process of longan fruits lacked systematic investigations. Our results showed that sucrose/hexose ratios and sugar receding rates of longan pulp varied among cultivars. Analysis of enzymes for sucrose synthesis and cleavage indicated that DlSAI showed the highest negative correlation with sucrose/hexose ratio at both of activity and expression level. Moreover, high SAI activity and DlSAI expression resulted in extremely low sucrose/hexose ratio in 'Luosanmu' longan from development to mature stages and a remarkable loss of sugar in 'Shixia' longan fruits during on-tree preservation. In conclusion, DlSAIs act as key factors influencing sucrose/hexose ratio and sugar receding through transcriptional and enzymatic regulations. These results might help improve the quality of on-tree preserved longan.

The Ubiquitin-Conjugating Enzyme Gene Family in Longan (Dimocarpus longan Lour.): Genome-Wide Identification and Gene Expression during Flower Induction and Abiotic Stress Responses.

Ubiquitin-conjugating enzymes (E2s or UBC enzymes) play vital roles in plant development and combat various biotic and abiotic stresses. Longan (Dimocarpus longan Lour.) is an important fruit tree in the subtropical region of Southeast Asia and Australia; however the characteristics of the UBC gene family in longan remain unknown. In this study, 40 D. longan UBC genes (DlUBCs), which were classified into 15 groups, were identified in the longan genome. An RNA-seq based analysis showed that DlUBCs showed distinct expression in nine longan tissues. Genome-wide RNA-seq and qRT-PCR based gene expression analysis revealed that 11 DlUBCs were up- or down-regualted in the cultivar "Sijimi" (SJ), suggesting that these genes may be important for flower induction. Finally, qRT-PCR analysis showed that the mRNA levels of 13 DlUBCs under SA (salicylic acid) treatment, seven under methyl jasmonate (MeJA) treatment, 27 under heat treatment, and 16 under cold treatment were up- or down-regulated, respectively. These results indicated that the DlUBCs may play important roles in responses to abiotic stresses. Taken together, our results provide a comprehensive insight into the organization, phylogeny, and expression patterns of the longan UBC genes, and therefore contribute to the greater understanding of their biological roles in longan.

The superoxide dismutase genes might be required for appropriate development of the ovule after fertilization in Xanthoceras sorbifolium.

KEY MESSAGE: Superoxide dismutase genes were expressed differentially along with developmental stages of fertilized ovules in Xanthoceras sorbifolium, and the XsMSD gene silencing resulted in the arrest of fertilized ovule development. A very small percentage of mature fruits (ca. 5%) are produced relative to the number of bisexual flowers in Xanthoceras sorbifolium because seeds and fruits are aborted at early stages of development after pollination. Reactive oxygen species (ROS) in plants are implicated in an extensive range of biological processes, such as programmed cell death and senescence. Superoxide dismutase (SOD) activity might be required to regulate ROS homeostasis in the fertilized ovules of X. sorbifolium. The present study identified five SOD genes and one SOD copper chaperone gene in the tree. Their transcripts were differentially expressed along different stages of fertilized ovule development. These genes showed maximum expression in the ovules at 3 days after pollination (DAP), a time point in which free nuclear endosperm and nucleus tissues rapidly develop. The XsCSD1, XsFSD1 and XsMSD contained seven, eight, and five introns, respectively. Analysis of the 5'-flanking region of XsFSD1 and XsMSD revealed many cis-acting regulatory elements. Evaluation of XsMSD gene function based on virus-induced gene silencing (VIGS) indicated that the gene was closely related to early development of the fertilized ovules and fruits. This study suggested that SOD genes might be closely associated with the fate of ovule development (aborted or viable) after fertilization in X. sorbifolium.

Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants.

Cloning and molecular characterization of phospholipase D (PLD) delta gene from longan (Dimocarpus longan Lour.).

Longan (Dimocarpus longan Lour.) is a non-climacteric fruit with a short postharvest life. The regulation of phospholipase D (PLD) activity closely relates to postharvest browning and senescence of longan fruit. In this study, a novel cDNA clone of longan PLDδ (LgPLDδ) was obtained and registered in GenBank (accession No. JF791814). The deduced amino acid sequence possessed all of the three typical domains of plant PLDs, a C2 domain and two catalytic HxKxxxxD motifs. The tertiary structure of LgPLDδ was further predicted. The western blot result showed that the LgPLDδ protein was specifically recognized by PLDδ antibody. The Q-RT-PCR (real-time quantitative PCR) result showed that the level of LgPLDδ mRNA expression was higher in senescent tissues than in developing tissues, which was also high in postharvest fruit. The western-blotting result further certified the different expression of LgPLDδ. These results provided a scientific basis for further investigating the mechanism of postharvest longan fruit adapting to environmental stress.

XsFAD2 gene encodes the enzyme responsible for the high linoleic acid content in oil accumulated in Xanthoceras sorbifolia seeds.

BACKGROUND: Xanthoceras sorbifolia Bunge is a valuable oilseed tree that has linoleic acid-rich seed oil. Microsomal oleate desaturase (FAD2; EC 1.3.1.35) is responsible for the conversion of oleic acid to linoleic acid during fatty acid synthesis. In this study, XsFAD2 was cloned from developing embryos of X. sorbifolia. RESULTS: XsFAD2 contained three histidine boxes, a C-terminal endoplasmic reticulum retrieval motif, and five putative transmembrane domains representing the characteristics of membrane-bound fatty acid desaturase. XsFAD2 expression in yeast cells resulted in linoleic acid (18:2) and palmitolinoleic acid (16:2) production, confirming the biological activity of the enzyme encoded by XsFAD2. These fatty acids are not normally present in wild-type yeast. Phylogenetic analysis indicated that XsFAD2 is located in a subgroup of FAD2 enzymes specifically or highly expressed in developing seeds. The expression level of XsFAD2 in seeds was much higher than those in leaves and petals. Furthermore, XsFAD2 expression pattern correlated well with linoleic acid accumulated in seeds. CONCLUSION: Results suggested that XsFAD2 is responsible for the high linoleic acid content in X. sorbifolia seed oil. This study provides insight on the regulation mechanism of fatty acid synthesis in X. sorbifolia seeds and a valuable gene for improving the oil quality in oilseed trees.

Two novel diacylglycerol acyltransferase genes from Xanthoceras sorbifolia are responsible for its seed oil content.

Xanthoceras sorbifolia is an excellent model system for studying triacylglycerol (TAG) biosynthesis in woody oilseed plants due to the high amount of seed oil, which is important for food and industrial uses. TAG is the major form of stored lipids in seeds and diacylglycerol acyltransferase (DGAT; EC 2. 3. 1. 20) catalyzes the final and critical step of TAG synthesis. Here, two novel DGAT genes, designated XsDGAT1 and XsDGAT2, were cloned from developing X. sorbifolia embryos. Sequence analysis showed that XsDGAT1 had little sequence homology to XsDGAT2. Heterologous expression of XsDGAT1 and XsDGAT2 in TAG-deficient yeast mutants restored TAG synthesis, confirming their biological activity. Expression of the two genes in wild-type Arabidopsis led to TAG synthesis and an increase in total seed oil in transgenic plants, with XsDGAT1 appearing to contribute to TAG synthesis at a greater level. Comparison of the expression patterns revealed that both XsDGAT1 and XsDGAT2 were expressed in the examined tissues and had similar spatiotemporal expression patterns with higher expression in embryos than in leaves and petals. The expression patterns of both XsDGAT1 and XsDGAT2 correlated with oil accumulation in developing X. sorbifolia embryos. These data suggest that XsDGAT1 and XsDGAT2 are both responsible for TAG synthesis in X. sorbifolia seeds.

Inhibitory effects of propyl gallate on tyrosinase and its application in controlling pericarp browning of harvested longan fruits.

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a key enzyme in pigment biosynthesis of organisms. The inhibitory effects of propyl gallate on the activity of mushroom tyrosinase and effects of propyl gallate on pericarp browning of harvested longan fruits in relation to phenolic metabolism were investigated. The results showed that propyl gallate could potently inhibit diphenolase activity of tyrosinase. The inhibitor concentration leading to 50% activity lost (IC50) was determined to be 0.685 mM. Kinetic analyses showed that propyl gallate was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (K(IS) and K(I)) were determined to be 2.135 and 0.661 mM, respectively. Furthermore, the results also showed that propyl gallate treatment inhibited activities of PPO and POD in pericarp of harvested longan fruits, and maintained higher contents of total phenol and flavonoid of longan pericarp. Moreover, propyl gallate treatment also delayed the increases of browning index and browning degree in pericarp of harvested longan fruits. Therefore, application of propyl gallate may be a promising method for inhibiting tyrosinase activity, controlling pericarp browning, and extending shelf life of harvested longan fruits.

Comparison on characterization of longan (Dimocarpus longan Lour.) polyphenoloxidase using endogenous and exogenous substrates.

Longan polyphenoloxidase (PPO) was extracted and partially purified using ammonium sulfate precipitation and dialysis. The PPO characterizations were compared using endogenous substrate (-)-epicatechin and exogenous substrate catechol. The optimal pH and optimal temperature for the PPO activity were different when reacting with both substrates. The addition of ethylenediaminetetraacetic acid disodium salt into both substrate-enzyme systems exhibited the same lowest inhibition of the PPO activity. L-ascorbic acid and L-cysteine were the best inhibitors to endogenous substrate-enzyme system, while L-ascorbic acid and glutathione were most effective inhibitors to exogenous substrate-enzyme system. Cupric (Cu2+), ferric (Fe3+), and ferrous (Fe2+) ions accelerated the enzymatic-catalyzed reactions of both substrates. Kinetic analysis indicated that longan PPO strongly bound endogenous substrate but possessed a higher catalytic efficiency to exogenous substrate, and moreover, (-)-epicatechin was determined as the optimal substrate for longan PPO. This study is useful to exactly illuminate the enzymatic-catalyzed browning mechanism of postharvest longan fruit.

Two endogenous substrates for polyphenoloxidase in pericarp tissues of postharvest rambutan fruit.

The catalytic oxidation of phenolic substrates by polyphenoloxidase (PPO) causes pericarp browning of postharvest rambutan fruit. In the present study, PPO and its endogenous substrates were extracted from rambutan pericarp tissues (RPT). The substrate extracts were sequentially partitioned with ethyl acetate and n-butanol. The analysis of total phenolic content showed that the most phenolic compounds were distributed in ethyl acetate fraction. By high-performance liquid chromatography (HPLC), (-)-epicatechin (EC) and proanthocyanidin A2 (PA2) were identified from this fraction. After reacting with rambutan PPO, EC turned brown rapidly within 10 min, indicating that it was a significant endogenous substrate. Although PA2 could also be oxidized by the PPO, it turned brown very slowly. In addition, because EC and PA2 were continually catalyzed into browning products by PPO during storage of the fruit at 4 and 25 degrees C, their contents in RPT gradually declined with the extended storage time. It was further observed that both substrate contents in rambutan fruit storing at 25 degrees C decreased more rapidly than that storing at 4 degrees C, suggesting that low temperature inhibited the catalytic oxidation of substrates so as to slow down pericarp browning. Practical Application: Pericarp browning is a serious problem to storage and transport of harvested rambutan fruit. A generally accepted opinion on the browning mechanism is the oxidation of phenolic substrates by PPO. Ascertaining PPO substrates will effectively help us to control enzymatic reaction by chemical methods so as to delay or even prevent pericarp browning of harvested rambutan fruit.

Rapid microwave-assisted transesterification of yellow horn oil to biodiesel using a heteropolyacid solid catalyst.

An efficient microwave-assisted transesterification (MAT) technique was developed to prepare biodiesel from yellow horn (Xanthoceras sorbifolia Bunge.) oil with a heteropolyacid (HPA) catalyst namely Cs(2.5)H(0.5)PW(12)O(40). A study for optimizing the reaction conditions such as reaction temperature, time, molar ratio of methanol/oil, catalyst amount, and recycle number of catalyst has been performed. The maximum yield of fatty acid methyl esters (FAMEs) reached 96.22% under optimal conditions of temperature 60 degrees C, 10 min, molar ratio of methanol/oil 12:1, 1% (w/w of oil) catalyst and minimum recycle number nine times. The final product of biodiesel, obtained after the new catalyzed process, was analyzed by gas chromatography. The results showed that the Cs(2.5)H(0.5)PW(12)O(40) heterogeneous acid catalyst had higher efficiency for transesterification under microwave irradiation compared with the conventional method. The product properties of yellow horn biodiesel are found to be in agreement with EN 14214 standard.

[The relationship between the desiccation-induced browning and the metabolism of active oxygen and phenolics in pericarp of postharvest longan fruit].

Longan (Dimocarpus longan Lour.) fruits are very susceptible to water loss and pericarp browning, and postharvest pericarp browning is the most important factors degrading the quality of longan fruit and shorting storage life. Pericarp browning has been attributed to desiccation, chilling, heat stress, senescence and pest or pathogen attack. Desiccation is the most main factor of induced-pericarp browning in longan. The relationship between water loss from pericarp and pericarp browning in longan cv. Fuyan fruits using open plastic punnets and sealed polyethylene bags at 10 degrees C +/-1 degrees C and 50% relative humidity, and the effect of pericarp water loss of the fruit on active oxygen metabolism and phenolics metabolism were investigated. Water loss resulted in rapid pericarp browning. Development of pericarp browning was higher with higher rate of water loss from pericarp and storage time (from 0 to 6 days). Water loss from pericarp was positively correlated with pericarp browning index significantly (P<0.01). Water loss from pericarp resulted in reduced activities of reactive-oxygen-scavenging enzymes (SOD, CAT, APX and GR), decreased amounts of endogenous antioxidant substances (AsA and GSH), and increased rates of O(-.)(2) production, MDA content and relative leakage rate, which showed that membrane structure was broken. Water loss from pericarp resulted in an increase in activity of PPO, and obvious reductions in total phenolic and flavonoid contents, whereas there was not obvious change in anthocyanin content. These results show that phenolics and flavonoids are the main substrates for PPO during desiccation-induced browning. Water loss from pericarp caused a significant increase in activity of POD, which also plays an important role in desiccation-induced browning in pericarp of longan fruit. Water loss from pericarp caused an increase in pH value, which resulted in changes in anthocyanin structure and color, the degradation of anthocyanin became easier. The results suggest that desiccation-induced browning of longan pericarp may be due to a decrease in activities of reactive-oxygen-scavenging enzymes and amounts of endogenous antioxidant substances, an accumulation of active oxygen, an increase in membrane lipid peroxidation, an injury of the integrity of cellular membrane structure, which, in turn, may cause cellular decompartmentation, resulted in PPO and POD, located in plastid and other organelle, to come into contact with phenolic and flavonoid substrates, located in vacuole, to form brown polymers.