Genome-Wide Identification and Expression Analysis of the Class III Peroxidase Gene Family under Abiotic Stresses in Litchi (Litchi chinensis Sonn.).

Class III peroxidases (CIII PRXs) are plant-specific enzymes with high activity that play key roles in the catalysis of oxidation-reduction reactions. In plants, CIII PRXs can reduce hydrogen peroxide to catalyze oxidation-reduction reactions, thereby affecting plant growth, development, and stress responses. To date, no systematic analysis of the CIII PRX gene family in litchi (Litchi chinensis Sonn.) has been documented, although the genome has been reported. In this study, a total of 77 CIII PRX (designated LcPRX) gene family members were predicted in the litchi genome to provide a reference for candidate genes in the responses to abiotic stresses during litchi growth and development. All of these LcPRX genes had different numbers of highly conserved PRX domains and were unevenly distributed across fifteen chromosomes. They were further clustered into eight clades using a phylogenetic tree, and almost every clade had its own unique gene structure and motif distribution. Collinearity analysis confirmed that there were eleven pairs of duplicate genes among the LcPRX members, and segmental duplication (SD) was the main driving force behind the LcPRX gene expansion. Tissue-specific expression profiles indicated that the expression levels of all the LcPRX family members in different tissues of the litchi tree were significantly divergent. After different abiotic stress treatments, quantitative real-time PCR (qRT-PCR) analysis revealed that the LcPRX genes responded to various stresses and displayed differential expression patterns. Physicochemical properties, transmembrane domains, subcellular localization, secondary structures, and cis-acting elements were also analyzed. These findings provide insights into the characteristics of the LcPRX gene family and give valuable information for further elucidating its molecular function and then enhancing abiotic stress tolerance in litchi through molecular breeding.

LcERF2 modulates cell wall metabolism by directly targeting a UDP-glucose-4-epimerase gene to regulate pedicel development and fruit abscission of litchi.

Elucidating the biochemical and molecular basis of premature abscission in fruit crops should help develop strategies to enhance fruit set and yield. Here, we report that LcERF2 contributes to differential abscission rates and responses to ethylene in Litchi chinensis (litchi). Reduced LcERF2 expression in litchi was observed to reduce fruit abscission, concurrent with enhanced pedicel growth and increased levels of hexoses, particularly galactose, as well as pectin abundance in the cell wall. Ecoptic expression of LcERF2 in Arabidopsis thaliana caused enhanced petal abscission, together with retarded plant growth and reduced pedicel galactose and pectin contents. Transcriptome analysis indicated that LcERF2 modulates the expression of genes involved in cell wall modification. Yeast one-hybrid, dual-luciferase reporter and electrophoretic mobility shift assays all demonstrated that a UDP-glucose-4-epimerase gene (LcUGE) was the direct downstream target of LcERF2. This result was further supported by a significant reduction in the expression of the A. thaliana homolog AtUGE2-4 in response to LcERF2 overexpression. Significantly reduced pedicel diameter and enhanced litchi fruit abscission were observed in response to LcUGE silencing. We conclude that LcERF2 mediates fruit abscission by orchestrating cell wall metabolism, and thus pedicel growth, in part by repressing the expression of LcUGE.

Expression of a Lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE with an Escherichia coli CYCLOPROPANE SYNTHASE Enhances Cyclopropane Fatty Acid Accumulation in Camelina Seeds.

Cyclopropane fatty acids (CPAs) are useful feedstocks for biofuels and bioproducts such as lubricants and biodiesel. Our goal is to identify factors that can facilitate the accumulation of CPA in seed triacylglycerol (TAG) storage oil. We hypothesized that the poor metabolism of CPA through the TAG biosynthetic network could be overcome by the addition of enzymes from species that naturally accumulate CPA in their seed oil, such as lychee (Litchi chinensis), which contains approximately 40% CPA in TAG. Our previous work on engineering CPA accumulation in crop and model plants identified a metabolic bottleneck between phosphatidylcholine (PC), the site of CPA biosynthesis, diacylglycerol (DAG), and TAG. Here, we report the cloning and heterologous expression in camelina (Camelina sativa) of a lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), which encodes the enzyme that catalyzes the transfer of the phosphocholine headgroup from PC to DAG. Camelina lines coexpressing LcPDCT and Escherichia coli CYCLOPROPANE SYNTHASE (EcCPS) showed up to a 50% increase of CPA in mature seed, relative to the EcCPS background. Stereospecific lipid compositional analysis showed that the expression of LcPDCT strongly reduced the level of C18:1 substrate at PC-sn-1 and PC-sn-2 (i.e. the sites of CPA synthesis), while the levels of CPA increased in PC-sn-2, DAG-sn-1 and DAG-sn-2, and both sn-1/3 and sn-2 positions in TAG. Taken together, these data suggest that the addition of PDCT facilitates more efficient movement of CPA from PC to DAG and establishes LcPDCT as a useful factor to combine with others to enhance CPA accumulation in plant seed oil.

Cyclopropane fatty acid biosynthesis in plants: phylogenetic and biochemical analysis of Litchi Kennedy pathway and acyl editing cycle genes.

KEY MESSAGE: This report describes the most extensive known gene discovery study from an oilseed that produces cyclopropane fatty acids, a novel industrial feedstock. Nature contains hundreds of examples of plant species that accumulate unusual fatty acids in seed triacylglycerols (TAG). Although lipid metabolic genes have been cloned from several exotic plant species, the underlying mechanisms that control the production of novel TAG species are still poorly understood. One such class of unusual fatty acids contain in-chain cyclopropane or cyclopropene functionalities that confer chemical and physical properties useful in the synthesis of lubricants, cosmetics, dyes, coatings, and other types of valuable industrial feedstocks. These cyclopropyl fatty acids, or CPFAs, are only produced by a small number of plants, primarily in the order Malvidae. Litchi chinensis is one member of this group; its seed oil contains at least 40 mol% CPFAs. Several genes, representing early, middle, and late steps in the Litchi fatty acid and TAG biosynthetic pathways have been cloned and characterized here. The tissue-specific and developmental transcript expression profiles and biochemical characteristics observed indicate which enzymes might play a larger role in Litchi seed TAG biosynthesis and accumulation. These data, therefore, provide insights into which genes likely represent the best targets for either silencing or overexpression, in future metabolic engineering strategies aimed at altering CPFA content.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn.

KEY MESSAGE: A novel LcGST4 was identified and characterized from Litchi chinensis . Expression and functional analysis demonstrated that it might function in anthocyanin accumulation in litchi. Glutathione S-transferases (GSTs) have been defined as detoxification enzymes for their ability to recognize reactive electrophilic xenobiotic molecules as well as endogenous secondary metabolites. Anthocyanins are among the few endogenous substrates of GSTs for vacuolar accumulation. The gene encoding a GST protein that is involved in anthocyanin sequestration from Litchi chinensis Sonn. has not been reported. Here, LcGST4, an anthocyanin-related GST, was identified and characterized. Phylogenetic analysis showed that LcGST4 was clustered with other known anthocyanin-related GSTs in the same clade. Expression analysis revealed that the expression pattern of LcGST4 was strongly correlated with anthocyanin accumulation in litchi. ABA- and light-responsive elements were found in the LcGST4 promoter, which is in agreement with the result that the expression of LcGST4 was induced by both ABA and debagging treatment. A GST activity assay in vitro verified that the LcGST4 protein shared universal activity with the GST family. Functional complementation of an Arabidopsis mutant tt19 demonstrated that LcGST4 might function in anthocyanin accumulation in litchi. Dual luciferase assay revealed that the expression of LcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchi.

LcINH1 as an inhibitor of cell wall invertase LcCWIN5 regulates early seed development in Litchi chinensis Sonn.

Sugar signal mediated by Cell wall invertase (CWIN) plays a central role in seed development. In higher plants, invertase inhibitors (INHs) suppress CWIN activities at a post-translational level. In Litchi chinensis cultivar 'Nuomici', impaired CWIN expression is associated with seed abortion. Here, the expression of LcINH1 was significantly higher in the funicle of seed-aborting cultivar 'Nuomici' than big-seeded cultivar 'Heiye'. Promoter analyses found LcINH1 contained a 404 bp repeat fragment with an endosperm regulatory element of Skn-1_motif. LcINH1 and LcCWIN2/5 were located in plasma membrane. LcINH1 was able to interact with LcCWIN5, but not with LcCWIN2. In vitro enzyme activity assay demonstrated that LcINH1 could inhibit CWIN activity. Silencing LcINH1 in 'Nuomici' resulted in normal seed development, paralleled increased CWIN activities and glucose levels. Transcriptome analysis identified 1079 differentially expressed genes (DEGs) in LcINH1-silenced fruits. KEGG analysis showed significant enrichment of DEGs in pathways related to transporters and plant hormone signal transduction. Weighted gene co-expression network analysis indicated that the turquoise module was highly correlated with fructose content, and LcSWEET3b was closely associated with early seed development. These findings suggest that LcINH1 regulate LcCWIN5 activity at the post-translational level to alter sucrose metabolism, thereby affecting early seed development in litchi.

Isolation of an abscisic acid senescence and ripening inducible gene from litchi and functional characterization under water stress.

A full-length abscisic acid (ABA) senescence and ripening inducible gene named LcAsr was obtained from litchi. Bioinformatic analysis showed that full-length LcAsr was 1,177 bp and contained an open reading frame (ORF) encoding 153 amino acids, 85- and 146-bp 5' and 3' UTRs, respectively. LcAsr was expressed in all organs, with preferential expression in the flower and low levels in pulp. The expression level of LcAsr in postharvest uncovered fruit reached a maximum at 24 h after harvest. When the litchi fruit was covered with plastic film, the LcAsr expression level remained constant. LcASR protein localized in the nucleus. LcAsr was transformed in Arabidopsis thaliana L. (ecotype Columbia) and four transgenic lines were obtained. One line, 35S::LcAsrD, was selected for drought tolerance analysis and showed higher tolerance to drought than the control. The activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase were much higher in the transgenic line than the control under drought conditions. The levels of several ABA/stress-regulated genes were investigated. The transcript level of responsive to ABA (RAB18) remained constant and responsive to dehydration (RD29A) displayed a slight decrease in the Columbia line (Col). However, the transcript levels of LcAsr, RAB18, and RD29A were greatly enhanced in the transgenic 35S::LcAsrD. The transcript levels of KAT1, KAT2, and SKOR were also markedly decreased in the transgenic line. These results suggest an important role of LcAsr as a protective molecule for water deficit and help to understand the molecular mechanism of postharvest litchi fruit dehydration.

The UDP glucose: flavonoid-3-O-glucosyltransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn.) during fruit coloration.

We analyzed a litchi cultivar that included three phenotypes for pericarp color, ranging from green, indicating the absence of anthocyanins, to yellow, and red. Anthocyanins, chlorophylls, carotenoids, and flavonoids were measured in the three stages. Fruit coloration of red-skinned litchi was mainly due to higher flavonols, and anthocyanin pigments, lower chlorophyll (higher chlorophyll degradation). Expression of four genes of the anthocyanin pathway coding for phenylalanine ammonialyase, chalcone synthase, flavanone-3-hydroxylase, and the UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), was analyzed by RT-PCR at three developmental stages from before the onset of ripening to full maturity. Gene expression patterns were compared to anthocyanin metabolites. The contents of anthocyanins and flavonols in the pericarps were consistent with the higher mRNA levels of UFGT, while, transcription of the other gene was not expected to follow the anthocyanin content. We suggest that UFGT might play an important role in anthocyanin biosynthesis in the pericarp of litchi. Thus, UFGT expression strongly influences fruit coloration in litchi.

Effects of Bacillus Subtilis CF-3 VOCs Combined with Heat Treatment on the Control of Monilinia fructicola in Peaches and Colletotrichum gloeosporioides in Litchi Fruit.

In order to study the effect of volatile organic compounds (VOCs) produced by Bacillus subtilis CF-3 combined with heat treatment on Monilinia fructicola in peach and Colletotrichum gloeosporioides in litchi fruit, fruits were treated with B. subtilis CF-3 VOCs and hot air alone or in combination. The quality indexes of peach and litchi fruit after treatment and the changes in defense-related enzymes were measured. The results showed that the B. subtilis CF-3 VOCs combined with heat treatment could significantly reduce the rot index of peach and litchi fruit, and effectively maintain firmness and soluble solids content, as well as reduce weight loss of fruits. The combined treatment effectively enhanced the activity of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) than either treatment alone, and enhanced the resistance of fruit to pathogenic fungi by activating disease-resistant enzymes (phenylalanine ammonia-lyase [PAL], chitinase [CHI], ß-1, 3-glucanase [GLU]) activity. In this study, B. subtilis CF-3 VOCs combined with heat treatment maintained the quality and delayed the decline of peach and litchi fruit, providing a theoretical basis for future applications. PRACTICAL APPLICATION: The combination of B. subtilis CF-3 VOCs and heat treatment reduce the extent of M. fructicola and C. gloeosporioides. The combination maintain the quality of peach and litchi better. The combination obviously improve the activity of defense-related enzyme in fruit.

Delay of Postharvest Browning in Litchi Fruit by Melatonin via the Enhancing of Antioxidative Processes and Oxidation Repair.

Melatonin acts as a crucial signaling and antioxidant molecule with multiple physiological functions in organisms. To explore effects of exogenous melatonin on postharvest browning and its possible mechanisms in litchi fruit, 'Ziniangxi' litchi fruits were treated with an aqueous solution of melatonin at 0.4 mM and then stored at 25 °C for 8 days. The results revealed that melatonin strongly suppressed pericarp browning and delayed discoloration during storage. Melatonin treatment reduced relative membrane-leakage rate and inhibited the generation of superoxide radicals (O2-·), hydrogen peroxide (H2O2), and malondialdehyde (MDA). Melatonin treatment markedly promoted the accumulation of endogenous melatonin; delayed loss of total phenolics, flavonoids, and anthocyanins; and enhanced the activities of antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). By contrast, the activities of browning-related enzymes including polyphenoloxidase (PPO, EC 1.10.3.1) and peroxidase (POD, EC 1.11.1.7) were reduced. In addition, melatonin treatment up-regulated the expression of four genes encoding enzymes for repair of oxidized proteins, including LcMsrA1, LcMsrA2, LcMsrB1, and LcMsB2. These findings indicate that the delay of pericarp browning and senescence by melatonin in harvested litchi fruit could be attributed to the maintenance of redox homeostasis by the improvement of the antioxidant capacity and modulation of the repair of oxidatively damaged proteins.

Characterization of polyphenol oxidase from litchi pericarp using (-)-epicatechin as substrate.

Polyphenol oxidase (PPO) from litchi (Litchi chinensis Sonn.) pericarp was characterized using (-)-epicatechin, which was the major endogenous polyphenol in litchi pericarp as a substrate. The optimum pH for PPO activity with (-)-epicatechin was 7.5, and the enzyme was unstable below pH 4.5 and stable in the pH range of 6.0-8.0. Residual activities of PPO were 86.25, 86.31, and 80.17% after 67 days of incubation at 4 degrees C at pH 6.0, 7.5, and 8.0, respectively. From thermostability studies, the Ki value increased with temperature and the results suggested that the enzyme was unstable above 45 degrees C. Moreover, the results also provided strong evidence that the denaturalization temperature of PPO was near 70 degrees C. The inhibition studies indicated that l-cysteine and glutathione were strong inhibitors even at low concentrations while NaF inhibited moderately. In addition, the results also indicated that the inhibition mechanisms of thiol groups were different from those of halide salts.

Degradation of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase.

The degradation mechanism of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase (PPO) was investigated using several model systems. The enzymically generated (-)-epicatechin o-quinone could induce cyanidin 3-rutinoside degradation. The results obtained in this study allowed us to propose a pathway for cyanidin 3-rutinoside degradation in the presence of (-)-epicatechin and litchi pericarp PPO. First, enzymatic oxidation of (-)-epicatechin produced the corresponding o-quinone, and then cyanidin 3-rutinoside and (-)-epicatechin competed for (-)-epicatechin o-quinone, resulting in degradation of cyanidin 3-rutinoside and regeneration of (-)-epicatechin. Moreover, the results of kinetic studies indicated this competition was influenced by both (-)-epicatechin concentration and cyanidin 3-rutinoside concentration in the model system.

[Changes in cell wall components and cell wall-degrading enzyme activities of postharvest longan fruit during aril breakdown].

Longan (Dimocarpus longan Lour.) fruits are very susceptible to pericarp browning and aril breakdown, and postharvest aril breakdown is one of the most important factors degrading the quality and shorting storage life of longan fruit. Changes in aril breakdown index, cell wall components and cell wall-degrading enzyme activities in aril of longan cv. Fuyan fruits using sealed polyethylene film bags (0.015 mm thick) at (10+/-1) degrees C were investigated. The main results were as follows. Development of aril breakdown was higher with storage time (from day 0 to day 36). Aril breakdown index was positively and significantly correlated with storage time (P<0.01). During development of aril breakdown, the total dry weight of the cell wall materials, protopectin, cellulose, semicellulose and cell wall protein contents of aril decreased progressively. The total dry weight of the cell wall materials, contents of protopectin, cellulose, semicellulose and cell wall protein of aril were all negatively correlated with aril breakdown index. There were low beta-galactosidase activity, and high activities of pectinesterase (PE), polygalacturonase (PG) and cellulase in aril of harvested fruit. PE activity in aril gradually decreased during development of aril breakdown. The activities of PG and cellulase in aril increased significantly during storage from day 6 to day 12 and from day 0 to day 12, respectively. The peaks enzyme activities of both PG and cellulase appeared on the 12th day after harvest, then the enzyme activity decreased; whereas, the activities of PE, PG and cellulase changed little from day 0 to day 24, and then rapidly decreased. The beta-galactosidase activity in aril decreased slightly during storage from day 0 to day 24. However, the beta-galactosidase activity increased significantly after day 24. Especially, the beta-galactosidase activity increased rapidly after 30 d of storage, in the meantime, the activities of PE, PG and cellulase almost disappeared. From the results it can be seen that the development of aril breakdown was due to the degradation of cell wall components such as protopectin, cellulose, semicellulose and cell wall protein. The early and middle phases of development of aril breakdown were mainly brought about by the action of PE, PG and cellulase, whereas, beta-galactosidase played the key role at the late phase of aril breakdown.

Differential expression of litchi XET genes in relation to fruit growth.

Xyloglucan endotransglycosylase (XET) catalyses the transglycosylation of xyloglucan, the major hemicellulose polymer, which has been thought to mediate the cross-linking of cellulose microfibrils in cellular walls and proposed to be involved in the control of cell wall relaxation. To understand the relationship between litchi fruit cracking and gene expression patterns, three XET genes from litchi fruit were identified and then examined for their expression profiles in pericarp and aril tissues at different development stages, using a cracking-resistant cultivar, 'Huaizhi', and a cracking-susceptible cultivar, 'Nuomici'. Three full-length cDNAs of 1267, 1095 and 1156 bp encoding XETs, named LcXET1, LcXET2 and LcXET3, respectively, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Northern blotting analysis showed that LcXET1 mRNA accumulation occurred much earlier in aril tissues at 59 days after anthesis (DAA) than in pericarp tissues at 73 DAA in 'Nuomici'. However, it appeared at almost the same time (66 DAA) in pericarp and aril tissues in 'Huaizhi', which suggested that differential accumulation of LcXET1 in pericarp and aril tissues in 'Nuomici' and 'Huaizhi' was closely associated with fruit cracking. LcXET2 mRNA accumulation could be detected in pericarp and aril tissues throughout fruit development but exhibited a differential accumulation pattern between pericarp and aril tissues. In the aril of 'Nuomici', intensive signal bands were detectable at 59-73 DAA in rapidly expanding fruits of 'Nuomici' but only weak bands could be found in the pericarp tissues. In contrast, moderate signal bands were detectable both in pericarp and aril tissues of 'Huaizhi' fruits. Furthermore, LcXET3 showed constitutive expression in both pericarp and aril tissues of developing 'Nuomici' and 'Huaizhi' litchi fruit. In addition, differential expression patterns of three XETs genes were observed in different tissues of litchi, with only LcXET1 being fruit-specific. To further address the role of LcXET in fruit cracking, alpha-naphthalene acetic acid (NAA) was used to treat 'Nuomoci' to reduce fruit cracking. Enhanced LcXET1 mRNA accumulation appeared in pericarp while LcXET2 and LcXET3 mRNA accumulation enhanced in aril tissues in the NAA-treated fruits. Thus, LcXET1 is more likely to play a role in reducing litchi fruit cracking than LcXET2 and LcXET3.

Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit.

Cloning and expression analysis of litchi (Litchi Chinensis Sonn.) polyphenol oxidase gene and relationship with postharvest pericarp browning.

Polyphenol oxidase (PPO) plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In this study, we cloned the litchi PPO gene (LcPPO, JF926153), and described its expression patterns. The LcPPO cDNA sequence was 2120 bps in length with an open reading frame (ORF) of 1800 bps. The ORF encoded a polypeptide with 599 amino acid residues, sharing high similarities with other plant PPO. The DNA sequence of the ORF contained a 215-bp intron. After carrying out quantitative RT-PCR, we proved that the LcPPO expression was tissue-specific, exhibiting the highest level in the flower and leaf. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits. Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe (the most rapid browning cultivar), but the lowest in Ziniangxi (the slowest browning cultivar) within 2 d postharvest. Preserving the fruits of Feizixiao in 0.2-µm plastic bag at room temperature would decrease the rate of pericarp water loss, delay the pericarp browning, and also cause the reduction of the pericarp PPO activity and LcPPO expression level within 3 d postharvest. In addition, postharvest storage of Feizixiao fruit stored at 4°C delayed the pericarp browning while decreasing the pericarp PPO activity and LcPPO expression level within 2 d after harvest. Thus, we concluded that the up-regulation of LcPPO expression in pericarp at early stage of postharvest storage likely enhanced the PPO activity and further accelerated the postharvest pericarp browning of litchi fruit.

Patterns of enzyme activities and gene expressions in sucrose metabolism in relation to sugar accumulation and composition in the aril of Litchi chinensis Sonn.

Sucrose metabolism enzymes, including invertase (EC 3.2.1.26), sucrose synthase (SS, EC 2.4.1.13), and sucrose phosphate synthase (SPS, EC 2.4.1.14), are key factors that determine fruit sugar accumulation and composition. Sugar concentration and sugar composition in the arils of 42 litchi cultivars were determined at maturity. The cultivars were grouped into three types according to their hexose/sucrose ratio. Five cultivars of each type were selected to monitor the activities and gene expressions of enzymes related to sucrose metabolism. Pattern changes in the arils of four cultivars with different sugar concentrations and compositions were traced from around 40 d after anthesis to full maturity. Highly significant positive correlations were observed between hexose/sucrose ratios and the activities and expression levels of soluble acid invertase (SAI) and SS among the 15 cultivars tested. The increase in hexose/sucrose ratio was accompanied by enhanced acid invertase (AI) and SS activities and the expression of their genes in Feizixiao (FZX) and Heiye (HY). By contrast, no significant correlation was observed between hexose/sucrose ratio and SPS. These results indicate that the sugar composition in litchi aril depends mainly on the sucrose cleavage enzymes AI and SS and not on the sucrose synthetic enzyme SPS. The cultivar Nuomici, which had the highest sugar content among the cultivars studied, displayed significantly lower activities of cell wall acid invertase, SAI, neutral invertase, and SS and lower expression levels of SAI and SS compared with HY, the cultivar with the lowest sugar content. The inconsistent patterns of sugar accumulation and activities and expressions of sucrose metabolism enzymes suggest that these sucrose metabolism enzymes are not necessarily related to sugar accumulation.

3-Hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) is highly associated with the cell division during the early stage of fruit development which determines the final fruit size in Litchi chinensis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.