Jianghui Xie

Transcriptome Profiling of Light-Regulated Anthocyanin Biosynthesis in the Pericarp of Litchi

Light is a key environmental factor that affects anthocyanin biosynthesis. To enhance our understanding of the mechanisms involved in light-regulated anthocyanin biosynthesis in the pericarp of litchi, we performed transcriptomic analyses on the basis of Illumina sequencing. Fruit clusters were bagged with double-layer Kraft paper bags at 42 days after anthesis. The bags were removed after 2 weeks. Under light conditions, anthocyanins accumulated rapidly in the pericarp. RNA sequences were de novo assembled into 75,935 unigenes with an average length of 913 bp. Approximately 74.5% of unigenes (56,601) were annotated against four public protein databases. A total of 16,622 unigenes that significantly differed in terms of abundance were identified. These unigenes are implicated in light signal perception and transduction, flavonoid biosynthesis, carotenoid biosynthesis, plant hormone signal transduction, and photosynthesis. In photoreceptors, the expression levels of UV RESISTANCE LOCUS 8 (UVR8), Phototropin 2 (PHOT2), Phytochrome B (PHYB), and Phytochrome C (PHYC) increased significantly when the fruits were exposed to light. This result indicated that they likely play important roles in anthocyanin biosynthesis regulation. After analyzed digital gene expression (DGE), we found that the light signal transduction elements of COP1 and COP10 might be responsible for anthocyanin biosynthesis regulation. After the bags were removed, nearly all structural and regulatory genes, such as UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), MYB, basic helix-loop-helix (bHLH), and WD40, involved in the anthocyanin biosynthetic pathway were upregulated. In addition to MYB-bHLH-WD40 transcription complex, ELONGATED HYPOCOTYL (HY5), NAM/ATAF/CUC (NAC), homeodomain leucine zipper proteins (ATHBs), and FAR-RED ELONGATED HYPOCOTYL (FHY) possibly participate in light-induced responses. On the basis of DGEs and qRT-PCR validation, we observed a light-induced anthocyanin biosynthesis and regulation pattern in litchi pericarp. This study enhanced our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in litchi pericarp.

Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion.

De Novo Assembly and Characterization of Pericarp Transcriptome and Identification of Candidate Genes Mediating Fruit Cracking in Litchi chinensis Sonn.

Fruit cracking has long been a topic of great concern for growers and researchers of litchi (Litchi chinensis Sonn.). To understand the molecular mechanisms underlying fruit cracking, high-throughput RNA sequencing (RNA-Seq) was first used for de novo assembly and characterization of the transcriptome of cracking pericarp of litchi. Comparative transcriptomic analyses were performed on non-cracking and cracking fruits. A total of approximately 26 million and 29 million high quality reads were obtained from the two groups of samples, and were assembled into 46,641 unigenes with an average length of 993 bp. These unigenes can be useful resources for future molecular studies of the pericarp in litchi. Furthermore, four genes (LcAQP, 1; LcPIP, 1; LcNIP, 1; LcSIP, 1) involved in water transport, five genes (LcKS, 2; LcGA2ox, 2; LcGID1, 1) involved in GA metabolism, 21 genes (LcCYP707A, 2; LcGT, 9; Lcβ-Glu, 6; LcPP2C, 2; LcABI1, 1; LcABI5, 1) involved in ABA metabolism, 13 genes (LcTPC, 1; Ca2+/H+ exchanger, 3; Ca2+-ATPase, 4; LcCDPK, 2; LcCBL, 3) involved in Ca transport and 24 genes (LcPG, 5; LcEG, 1; LcPE, 3; LcEXP, 5; Lcβ-Gal, 9; LcXET, 1) involved in cell wall metabolism were identified as genes that are differentially expressed in cracked fruits compared to non-cracked fruits. Our results open new doors to further understand the molecular mechanisms behind fruit cracking in litchi and other fruits, especially Sapindaceae plants.

Identification and evaluation of two diagnostic markers linked to Fusarium wilt resistance (race 4) in banana (Musa spp.).

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (FOC4) results in vascular tissue damage and ultimately death of banana (Musa spp.) plants. Somaclonal variants of in vitro micropropagated banana can hamper success in propagation of genotypes resistant to FOC4. Early identification of FOC4 resistance in micropropagated banana plantlets is difficult, however. In this study, we identified sequence-characterized amplified region (SCAR) markers of banana associated with resistance to FOC4. Using pooled DNA from resistant or susceptible genotypes and 500 arbitrary 10-mer oligonucleotide primers, 24 random amplified polymorphic DNA (RAPD) products were identified. Two of these RAPD markers were successfully converted to SCAR markers, called ScaU1001 (GenBank accession number HQ613949) and ScaS0901 (GenBank accession number HQ613950). ScaS0901 and ScaU1001 could be amplified in FOC4-resistant banana genotypes (“Williams 8818-1” and Goldfinger), but not in five tested banana cultivars susceptible to FOC4. The two SCAR markers were then used to identify a somaclonal variant of the genotype “Williams 8818-1”, which lost resistance to FOC4. Hence, the identified SCAR markers can be applied for a rapid quality control of FOC4-resistant banana plantlets immediately after the in vitro micropropagation stage. Furthermore, ScaU1001 and ScaS0901 will facilitate marker-assisted selection of new banana cultivars resistant to FOC4.

Functional properties of a cysteine proteinase from pineapple fruit with improved resistance to fungal pathogens in Arabidopsis thaliana.

In plant cells, many cysteine proteinases (CPs) are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L.) belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps), and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3). Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

The banana E2 gene family: Genomic identification, characterization, expression profiling analysis.

The E2 is at the center of a cascade of Ub1 transfers, and it links activation of the Ub1 by E1 to its eventual E3-catalyzed attachment to substrate. Although the genome-wide analysis of this family has been performed in some species, little is known about analysis of E2 genes in banana. In this study, 74 E2 genes of banana were identified and phylogenetically clustered into thirteen subgroups. The predicted banana E2 genes were distributed across all 11 chromosomes at different densities. Additionally, the E2 domain, gene structure and motif compositions were analyzed. The expression of all of the banana E2 genes was analyzed in the root, stem, leaf, flower organs, five stages of fruit development and under abiotic stresses. All of the banana E2 genes, with the exception of few genes in each group, were expressed in at least one of the organs and fruit developments, which indicated that the E2 genes might involve in various aspects of the physiological and developmental processes of the banana. Quantitative RT-PCR (qRT-PCR) analysis identified that 45 E2s under drought and 33 E2s under salt were induced. To the best of our knowledge, this report describes the first genome-wide analysis of the banana E2 gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family.

Transcriptome analysis of ‘Sijihua’ longan (Dimocarpus longan L.) based on next-generation sequencing technology

ABSTRACT ‘Sijihua’ longan (Dimocarpus longan Lour.) blossoms throughout the year, and hence it is a useful material for studies on the mechanism of flowering. However, few genomic resources such as DNA sequences or markers are currently available for ‘Sijihua’ longan. To increase our understanding of the genetic control of important flowering characteristics in ‘Sijihua’ longan, we constructed a mixed cDNA library of ‘Sijihua’ longan using Illumina paired-end sequencing technology and obtained a total of 90,069,246 high-quality reads. Among the unigenes, 43.16% (59,588) had at least one BLASTX hit against the Arabidopsis thaliana sequence database and 42.83% (59,130) had significant similarity with proteins in the NCBI non-redundant protein database. Of these annotated unigenes, a total of 15,956 (11.56%) were mapped onto 271 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. Furthermore, 11,402 unigenes were converted into 13,236 potential simple sequence repeats (SSRs). Dinucleotide SSRs were the dominant repeat motif, with a frequency of 54.3%, followed by trinucleotide (34.6%) and tetranucleotide (4.7%) repeat motifs. AG/CT repeats (46.29%) were the most frequent motifs in the data set. We focused on gene discovery in the area of regulation of flowering, and transcripts were identified for almost every gene involved in the control of flowering. This study demonstrates that Illumina sequencing technology is a rapid and informative approach to gene discovery and molecular marker development in non-model organisms without a whole genome database. Our results provide a comprehensive sequence resource for investigating the mechanisms of flowering in ‘Sijihua’ longan, and will establish a platform for further studies on longan.

Disease resistance of ‘Zill’ and ‘Keitt’ mango fruit to anthracnose in relation to defence enzyme activities and the content of anti-fungal substances

Summary Mango (Magnifera indica L.) is a popular fruit, but it is susceptible to post-harvest diseases. To study the resistance of mango fruit to anthracnose disease and the possible defence mechanisms involved, the resistance of two mango cultivars (‘Zill’ and ‘Keitt’) was examined in relation to their defence enzyme systems. The results showed that in both young or commercially mature fruit inoculated with Colletotrichum gloeosporioides, lesion sizes on ‘Keitt’ fruit were smaller than on ‘Zill’ fruit. For non-inoculated fruit harvested at commercial maturity, the disease index of ‘Keitt’ was lower than that of ‘Zill’. This indicates that ‘Keitt’ was more disease resistant than ‘Zill’. Compared with ‘Zill’, ‘Keitt’ fruit showed higher phenylalanine ammonia lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) activities during development and storage, as well as elevated levels of H2O2, total phenolic compounds and lignin during development, and higher contents of H2O2 and lignin in harvested fruit early during storage. These results suggest that defence enzymes and compounds plays important roles in the resistance of mango fruit to anthracnose disease and could be used as indicators to screen for mango cultivars that are more resistant to post-harvest diseases.

Effects of 1-MCP on oxidative parameters and quality in ‘Pearl’ guava (Psidium guajava L.) fruit

Summary Guava (Psidium guajava L. ‘Pearl’) fruit were harvested at approx. the 80% mature stage. Fruit were exposed to 1.0 µl l–1 1-methylcyclopropene (1-MCP) gaseous vapour for 12 h in a closed chamber maintained at 25ºC, stored at 28ºC and 60 – 65% relative humidity (RH) for 15 d, then assessed for their physiological and quality characteristics. The results showed that 1-MCP treatment delayed any increase in the incidence of decay, weight loss, and malondialdehyde (MDA) content. 1-MCP treatment had a positive effect on the retention of skin colour and on the firmness of guava fruit. Meanwhile, 1-MCP treatment maintained a higher vitamin C content and a lower soluble solids content, but titratable acidity was not affected by 1-MCP treatment. Moreover, 1-MCP treatment increased the activities of superoxide dismutase, catalase, and peroxidase, while reducing the activity of polyphenol oxidase. These results suggest that 1-MCP could delay the ripening process, maintain quality, and increase the anti-oxidant capacity of guava fruit stored at 28ºC.

Novel pH control strategy for glutathione overproduction in batch cultivation of Candida utilis

The effects of pH values on cell growth and glutathione (GSH) production were studied in batch cultivation of Candida utilis. According to the fact that lower pH value favors cells growth but retards GSH production and higher pH value promotes GSH production while inhibits cells growth, a pH-shift strategy, optimized via simulating Gauss function, was developed. By applying two-stage pH-shift strategy of controlling pH at 5.0 for first 7.5 h and switching to 6.0 afterwards, final GSH yield and productivity reached 279 and 12.7 mg/l/h after 22 h cultivation, increased by 30 and 42%, respectively, compared to constant pH 5.5 operation. Moreover, by feeding glucose instead of sulphuric acid (H2SO4) solution to control pH, maximal GSH yield of 315 mg/l was achieved, suggesting application of pH-shift strategy for GSH overproduction as being feasible.