Shaohui Yang

Genome-wide identification, characterization and expression analysis of populus

BackgroundLeucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species.ResultsIn this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments.ConclusionsThis study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion of this gene family. Populus and Arabidopsis LRR-RLK homologues not only share similar genetic structures but also exhibit comparable expression patterns which point to the possible functional conservation of these LRR-RLKs in two model systems. Transcriptome profiling provides the first insight into the functional divergence among PtLRR-RLK gene subfamilies and suggests that they might take important roles in growth and adaptation of tree species.

Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes

BackgroundLeucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species.ResultsIn this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments.ConclusionsThis study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion of this gene family. Populus and Arabidopsis LRR-RLK homologues not only share similar genetic structures but also exhibit comparable expression patterns which point to the possible functional conservation of these LRR-RLKs in two model systems. Transcriptome profiling provides the first insight into the functional divergence among PtLRR-RLK gene subfamilies and suggests that they might take important roles in growth and adaptation of tree species.

Antioxidative, antibrowning and antibacterial activities of sixteen floral honeys

Commonly consumed honeys from sixteen different single floral sources were analyzed for their in vitro antioxidant capacities by several methods including DPPH, ABTS, FRAP, SASR and MDA assays. The total polyphenol contents varied among the tested honeys and were highly correlated to their antioxidant capacity values. The antioxidant capacity of Chinese milk vetch flower honeys was significantly higher than those of other flower honeys. All honeys tested were active in inhibiting the browning of apple homogenate and linden honey displayed the highest inhibition rate as 85%. When the antimicrobial activity of the investigated honeys was screened using Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli), clover honey exhibited the strongest antibacterial activity as 2.2 mg mL(-1) kanamycin equivalent inhibition.

Impact of heavy metal stresses on the growth and auxin homeostasis of Arabidopsis seedlings

The phytohormone auxin is an essential mediator in many aspects of plant development. Its dynamic and differential distribution within the plant is regulated by a variety of environmental cues including heavy metal stimuli. In the present study, we first evaluated the toxic effects of seven heavy metals including Pb2+, Cd2+, Hg2+, Ni2+. Zn2+, Co2+ and Cu2+ in their excess on the model plant, Arabidopsis thaliana. Various morphological defects including loss in fresh weight and leaf area, decrease of the primary root length and stimulation of the lateral root density occurred to a different extent among seven heavy metals. Next, using an indicative DR5:GUS reporter line of Arabidopsis, the auxin accumulation and distribution within plant seedlings were found to be dramatically and differentially affected by these heavy metals. We further analyzed the transcriptional changes of 27 selected auxin homeostasis-related genes by qRT-PCR technique and found that upon various heavy metals, the expressions of the candidate genes were distinctly altered in shoots and roots. Our data indicated that when confronted with excessive heavy metals, plants could dynamically and differentially regulate the transcription of auxin-related genes to adjust the location and effective accumulation of auxin within the plant for better adaptation and survival under the adverse environment.

Genome-wide characterization, expression and functional analysis of CLV3/ESR gene family in tomato

BackgroundBy encoding a group of small secretory peptides, the members of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) family play important roles in cell-to-cell communication to control the balance between stem cell proliferation and differentiation in plant development. Despite recent identification and characterization of members of this gene family in several plant species, little is known about its functional role in plants with fleshy fruits.ResultsIn total, fifteen CLE genes (SlCLE1-15) were identified from tomato (Solanum lycopersicum cv. ‘Heinz-1706’) genome and their multiple characters including phylogeny, gene structures, chromosome locations, conserved motifs and cis-elements in the promoter sequences, were analyzed. Real-time PCR analysis showed that 13 out of 15 identified SlCLE genes are transcribed and exhibit remarkably unique expression patterns among tissues and organs. In particular, SlCLE12, the homologue of Arabidopsis CLE41/44 gene, appears to be the dominant CLE gene in most of tested tissues with its maximum expression found in vascular tissues. Meanwhile, SlCLE1, 10, 13 exhibit specific but distinct expression in flower bud, root and shoot apex, respectively. More notably, several SlCLEs are dramatically regulated in their transcriptional levels during fruit development and ripening, indicating significant role these genes may potentially play in the critical physiological process. Upon the treatment with synthetic peptides corresponding to the 12-aa CLE domains of SlCLE 10, 12 and 13, tomato seedlings exhibit a clear reduction in root length to varying degrees.ConclusionsThis study provides a comprehensive genomic analysis of CLE gene family in tomato, a crop species with fleshy fruit. Differential expression patterns of various SlCLEs provide important insights into the functional divergence of CLE signaling cascade in Solanaceae species, especially their potential involvements in the regulation of fruit development and ripening.

Vascular expression of Populus LRR-RLK genes and the effects of their overexpression on wood formation

Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases (RLKs) in land plants. Although some LRR-RLKs have been characterized as playing key roles in cell-to-cell communications, the physiological roles of most LRR-RLKs remain to be determined. In this study, we first screened the Populus trichocarpa expressed sequence tag and microarray database to look for PtLRR-RLKs differentially and preferentially expressed across the vascular cambial zone. The cell-specific expression of seven selected PtLRR-RLK genes was then verified by the GUS (β-glucuronidase) reporter gene assay. Nearly all of the seven types of plants transformed with PtLRR-RLKpro:GUS fusions showed a high level of transcription in the developing primary xylem and interfascicular cambium during the early stages of xylem differentiation. In stems undergoing secondary growth, the GUS activities driven by the seven PtLRR-RLK promoters were mainly associated with the cambial zone. These seven PtLRR-RLK genes were then individually overexpressed in hybrid poplars, and most transgenic poplars did not exhibit significant phenotypic changes. However, overexpression of PtLRR-RLK1, the putative ortholog of TDR/PXY in Arabidopsis, led to an ectopic accumulation of lignin in the pith along with an enlarged secondary xylem and increased lignin content without perceptible effect on secondary cell wall thickness, which made this transgene a likely candidate as a transducer of signals during plant secondary growth and wood formation.

Gain-of-function analysis of poplar CLE genes in Arabidopsis by exogenous application and over-expression assays

Among 50 CLE gene family members in the Populus trichocarpa genome, three and six PtCLE genes encode a CLE motif sequence highly homologous to Arabidopsis CLV3 and TDIF peptides, respectively, which potentially make them functional equivalents. To test and compare their biological activity, we first chemically synthesized each dodecapeptide and analysed itsi n vitro bioactivity on Arabidopsis seedlings. Similarly, but to a different extent, three types of poplar CLV3-related peptides caused root meristem consumption, phyllotaxis disorder, anthocyanin accumulation and failure to enter the bolting stage. In comparison, application of two poplar TDIF-related peptides led to root length promotion in a dose-dependent manner with an even stronger effect observed for poplar TDIF-like peptide than TDIF. Next, we constructed CaMV35S:PtCLE transgenic plants for each of the nine PtCLE genes. Phenotypic abnormalities exemplified by arrested shoot apical meristem and abnormal flower structure were found to be more dominant and severe in 35S:PtCLV3 and 35S:PtCLV3-like2 lines than in the 35S:PtCLV3-like line. Disordered vasculature was detected in both stem and hypocotyl cross-sections in Arabidopsis plants over-expressing poplar TDIF-related genes with the most defective vascular patterning observed for TDIF2 and two TDIF-like genes. Phenotypic difference consistently observed in peptide application assay and transgenic analysis indicated the functional diversity of nine poplar PtCLE genes under investigation. This work represents the first report on the functional analysis of CLE genes in a tree species and constitutes a basis for further study of the CLE peptide signalling pathway in tree development.

Two Groups of Thellungiella salsuginea RAVs Exhibit Distinct Responses and Sensitivity to Salt and ABA in Transgenic Arabidopsis

Containing both AP2 domain and B3 domain, RAV (Related to ABI3/VP1) transcription factors are involved in diverse functions in higher plants. A total of eight TsRAV genes were isolated from the genome of Thellungiella salsuginea and could be divided into two groups (A- and B-group) based on their sequence similarity. The mRNA abundance of all Thellungiella salsuginea TsRAVs followed a gradual decline during seed germination. In Thellungiella salsuginea seedling, transcripts of TsRAVs in the group A (A-TsRAVs) were gradually and moderately reduced by salt treatment but rapidly and severely repressed by ABA treatment. In comparison, with a barely detectable constitutive expression, the transcriptional level of TsRAVs in the group B (B-TsRAVs) exhibited a moderate induction in cotyledons when confronted with ABA. We then produced the “gain-of-function” transgenic Arabidopsis plants for each TsRAV gene and found that only 35S:A-TsRAVs showed weak growth retardation including reduced root elongation, suggesting their roles in negatively controlling plant growth. Under normal conditions, the germination process of all TsRAVs overexpressing transgenic seeds was inhibited with a stronger effect observed in 35S:A-TsRAVs seeds than in 35S:B-TsRAVs seeds. With the presence of NaCl, seed germination and seedling root elongation of all plants including wild type and 35S:TsRAVs plants were retarded and a more severe inhibition occurred to the 35S:A-TsRAV transgenic plants. ABA treatment only negatively affected the germination rates of 35S:A-TsRAV transgenic seeds but not those of 35S:B-TsRAV transgenic seeds. All 35S:TsRAVs transgenic plants showed a similar degree of reduction in root growth compared with untreated seedlings in the presence of ABA. Furthermore, the cotyledon greening/expansion was more severely inhibited 35S:A-TsRAVs than in 35S:B-TsRAVs seedlings. Upon water deficiency, with a wider opening of stomata, 35S:A-TsRAVs plants experienced a faster transpirational water loss than wild type and 35S:B-TsRAVs lines. Taken together, our results suggest that two groups of TsRAVs perform distinct regulating roles during plant growth and abiotic defense including drought and salt, and A-TsRAVs are more likely than B-TsRAVs to act as negative regulators in the above-mentioned biological processes.

Overexpression of poplar wounding-inducible genes in Arabidopsis caused improved resistance against Helicoverpa armigera (Hübner) larvae.

Four highly inducible genes of poplar trees, PtdKTI5, PtdWIN4, PtdPOP3 from hybrid poplar (Populus trichocarpa × P. deltoides) and PtKTI2 from trembling aspen (Populus tremuloides Michx.) have been individually transformed into Arabidopsis thaliana for overexpression. High transcriptional level of each transgene in transgenic Arabidopsis lines was confirmed by RT-PCR analysis. The development, body weight and survivorship of cotton bollworm (Helicoverpa armigera) fed on four types of transgenic Arabidopis plants were evaluated in the laboratory. Our data indicated that these four Populus defense-related genes exhibited various degree of insectital activity on larval and postlarval development of cotton bollworm and may be utilized for herbivore resistance improvement in plant genetic engineering.

Truncated BAM receptors interfere the apical meristematic activity in a dominant negative manner when ectopically expressed in Arabidopsis

Small, secreted signaling peptides that are perceived by receptor-like kinases (RLKs) constitute an important regulatory mechanism in plant organ formation and stem cell maintenance. However, functional redundancy at the level of both ligand and receptor families often makes it difficult to clearly discern the role of individual members by a genetic approach. Here, we show that driven by a constitutive CaMV 35S promoter, a truncated BAM protein (BAMΔ) that lacks either the signal peptide (SP) or the cytoplasmic kinase (Ki) domain could cause defective shoot apical meristem (SAM) maintenance, which phenotypically resembled the triple bam mutant. Such a dominant-negative effect could also be achieved when the same transgene was driven by the native AtBAM1 promoter, but not by the CLV1 promoter. When introduced into a clv1-4 background, BAMΔ proteins abolished the typical clv phenotype by suppressing the transcriptional level of clv1-4. In addition to a clear reduction in root length and a decreased number of meristematic cells, the 35S:BAMΔ transgenic seedlings exhibited considerable resistance to CLE40p- but not to CLV3p-mediated root growth inhibition, implying that BAMs play key roles in the regulation of proximal meristem activity in root through CLE40 peptide. Findings present here not only provide evidence that truncated BAM proteins are strongly dominant negative in regulating apical meristem development but also propose that expression of a truncated version of plant LRR receptor kinase could potentially be used as a powerful tool to reveal its in vivo function in signal transduction.