Xin-Rong Ma

Comparative transcriptome analysis of tomato (Solanum lycopersicum) in response to exogenous abscisic acid

BackgroundAbscisic acid (ABA) can regulate the expressions of many stress-responsive genes in plants. However, in defense responses to pathogens, mounting evidence suggests that ABA plays variable roles. Little information exists about genome-wide gene expression in ABA responses in tomato (Solanum lycopersicum L.), a model fruit crop plant.ResultsGlobal transcriptome profiles of tomato leaf responses to exogenous ABA were generated using Illumina RNA-sequencing. More than 173 million base pair reads were mapped onto the tomato reference genome and the expression pattern differences between treated and control leaves were assessed. In total, 50,616 transcripts were generated. Among them, 42,583 were functionally annotated in the NCBI non-redundant database and 47,877 in the tomato genome reference. Additionally, 31,107 transcripts were categorized into 57 functional groups based on Gene Ontology terms, and 14,371 were assigned to 310 Kyoto Encyclopedia of Genes and Genomes pathways. In both the ABA treatment and control samples, 39,671 transcripts were available to analyze their expressions, of which 21,712 (54.73%) responded to exogenous ABA. Of these transcripts, 2,787 were significantly differently expressed genes (DEGs). Many known and novel ABA-induced and -repressed genes were found. Exogenous ABA can influence the ABA signaling pathway with PYR/PYL/RCARs-PP2Cs-SnRK2s as the center. Eighteen PYL genes were detected. A large number of genes related to various transcription factors, heat shock proteins, pathogen resistance, and the salicylic acid, jasmonic acid, and ethylene signaling pathways were up-regulated by exogenous ABA.ConclusionsThe results indicated that ABA has the potential to improve pathogen-resistance and abiotic stress tolerance in tomato. This study presents the global expression analysis of ABA-regulated transcripts in tomato and provides a robust database for investigating the functions of genes induced by ABA.

Microbial community and removal of nitrogen via the addition of a carrier in a pilot-scale duckweed-based wastewater treatment system

Carriers were added to a pilot-scale duckweed-based (Lemna japonica 0223) wastewater treatment system to immobilize and enhance microorganisms. This system and another parallel duckweed system without carriers were operated for 1.5 years. The results indicated the addition of the carrier did not significantly affect the growth and composition of duckweed, the recovery of total nitrogen (TN), total phosphorus (TP) and CO2 or the removal of TP. However, it significantly improved the removal efficiency of TN and NH4(+)-N (by 19.97% and 15.02%, respectively). The use of 454 pyrosequencing revealed large differences of the microbial communities between the different components within a system and similarities within the same components between the two systems. The carrier biofilm had the highest bacterial diversity and relative abundance of nitrifying bacteria (3%) and denitrifying bacteria (24% of Rhodocyclaceae), which improved nitrogen removal of the system. An efficient N-removal duckweed system with enhanced microorganisms was established.

Plant programmed cell death caused by an autoactive form of Prf is suppressed by co-expression of the Prf LRR domain.

In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.

Heat Shock Factor Genes of Tall Fescue and Perennial Ryegrass in Response to Temperature Stress by RNA-Seq Analysis

Heat shock factors (Hsfs) are important regulators of stress-response in plants. However, our understanding of Hsf genes and their responses to temperature stresses in two Pooideae cool-season grasses, Festuca arundinacea, and Lolium perenne, is limited. Here we conducted comparative transcriptome analyses of plant leaves exposed to heat or cold stress for 10 h. Approximately, 30% and 25% of the genes expressed in the two species showed significant changes under heat and cold stress, respectively, including subsets of Hsfs and their target genes. We uncovered 74 Hsfs in F. arundinacea and 52 Hsfs in L. perenne, and categorized these genes into three subfamilies, HsfA, HsfB, and HsfC based on protein sequence homology to known Hsf members in model organisms. The Hsfs showed a strong response to heat and/or cold stress. The expression of HsfAs was elevated under heat stress, especially in class HsfA2, which exhibited the most dramatic responses. HsfBs were upregulated by the both temperature conditions, and HsfCs mainly showed an increase in expression under cold stress. The target genes of Hsfs, such as heat shock protein (HSP), ascorbate peroxidase (APX), inositol-3-phosphate synthase (IPS), and galactinol synthase (GOLS1), showed strong and unique responses to different stressors. We comprehensively detected Hsfs and their target genes in F. arundinacea and L. perenne, providing a foundation for future gene function studies and genetic engineering to improve stress tolerance in grasses and other crops.

The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans.

Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries.

Using proteomic analysis to investigate uniconazole-induced phytohormone variation and starch accumulation in duckweed (Landoltia punctata).

BackgroundDuckweed (Landoltia punctata) has the potential to remediate wastewater and accumulate enormous amounts of starch for bioethanol production. Using systematical screening, we determined that the highest biomass and starch percentage of duckweed was obtained after uniconazole application. Uniconazole contributes to starch accumulation of duckweed, but the molecular mechanism is still unclear.ResultsTo elucidate the mechanisms of high starch accumulation, in the study, the responses of L. punctata to uniconazole were investigated using a quantitative proteomic approach combined with physiological and biochemical analysis. A total of 3327 proteins were identified. Among these identified proteins, a large number of enzymes involved in endogenous hormone synthetic and starch metabolic pathways were affected. Notably, most of the enzymes involved in abscisic acid (ABA) biosynthesis showed up-regulated expression, which was consistent with the content variation. The increased endogenous ABA may up-regulate expression of ADP-glucose pyrophosphorylase to promote starch biosynthesis. Importantly, the expression levels of several key enzymes in the starch biosynthetic pathway were up-regulated, which supported the enzymatic assay results and may explain why there is increased starch accumulation.ConclusionsThese generated data linked uniconazole with changes in expression of enzymes involved in hormone biosynthesis and starch metabolic pathways and elucidated the effect of hormones on starch accumulation. Thus, this study not only provided insights into the molecular mechanisms of uniconazole-induced hormone variation and starch accumulation but also highlighted the potential for duckweed to be feedstock for biofuel as well as for sewage treatment.

Cloning and characterization of farnesyl pyphosphate synthase gene from the ABA-producing fungi Botrytis cinerea.

Farnesyl pyphosphate synthase (FPPS) catalyzes the systhesis of farnesyl pyphosphate and appears to be a promising regulation site of Abscisic acid (ABA) biosynthesis pathway in fungi. Here we reported the isolation and characterization of Botrytis cinerea (FPPS) gene. The cloned FPPS gene carries an open reading frame of 1044-bp encoding a deduced protein of 347 amino acids with a molecular weight of 39.83 kDa, and the coding region is interrupted with a 63-bp intron. Comparison analysis showed that the deduced amino acids sequence share high similarity with other known FPPS gene. Southern blot revealed a single copy of FPPS gene in the genomic DNA. The result of transcription analysis indicated that the cloned FPPS gene expressed constitutively and was not induced in ABA accumulation phase.

Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.