Tong Yuan

BAK1 and BKK1 Regulate Brassinosteroid-Dependent Growth and Brassinosteroid-Independent Cell-Death Pathways

Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.

Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

BackgroundTransmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated.ResultsAs a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community.ConclusionsMost of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

GeoChip 4: a functional gene‐array‐based high‐throughput environmental technology for microbial community analysis

Micro‐organisms play critical roles in many important biogeochemical processes in the Earths biosphere. However, understanding and characterizing the functional capacity of microbial communities are still difficult due to the extremely diverse and often uncultivable nature of most micro‐organisms. In this study, we developed a new functional gene array, GeoChip 4, for analysing the functional diversity, composition, structure, metabolic potential/activity and dynamics of microbial communities. GeoChip 4 contained approximately 82 000 probes covering 141 995 coding sequences from 410 functional gene families related to microbial carbon (C), nitrogen (N), sulphur (S), and phosphorus (P) cycling, energy metabolism, antibiotic resistance, metal resistance/reduction, organic remediation, stress responses, bacteriophage and virulence. A total of 173 archaeal, 4138 bacterial, 404 eukaryotic and 252 viral strains were targeted, providing the ability to analyse targeted functional gene families of micro‐organisms included in all four domains. Experimental assessment using different amounts of DNA suggested that as little as 500 ng environmental DNA was required for good hybridization, and the signal intensities detected were well correlated with the DNA amount used. GeoChip 4 was then applied to study the effect of long‐term warming on soil microbial communities at a Central Oklahoma site, with results indicating that microbial communities respond to long‐term warming by enriching carbon degradation, nutrient cycling (nitrogen and phosphorous) and stress response gene families. To the best of our knowledge, GeoChip 4 is the most comprehensive functional gene array for microbial community analysis.

Response of an allergenic species, Ambrosia psilostachya (Asteraceae), to experimental warming and clipping: Implications for public health

We examined the responses of an allergenic species, western ragweed (Ambrosia psilostachya DC.), to experimental warming and clipping. The experiment was conducted in a tallgrass prairie in Oklahoma, USA, between 1999 and 2001. Warming increased ragweed stems by 88% when not clipped and 46% when clipped. Clipping increased ragweed stems by 75% and 36% in the control and warmed plots, respectively. In 2001, warming resulted in a 105% increase in ragweed aboveground biomass (AGB), and the ratio of ragweed AGB to total AGB increased by 79%. Dry mass per ragweed stem in the warmed plots was 37% and 38% greater than that in the control plots in 2000 and 2001, respectively. Although warming caused no difference in pollen production per stem, total pollen production increased by 84% (P < 0.05) because there were more ragweed stems. Experimental warming significantly increased pollen diameter from 21.2 μm in the control plots to 23.9 μm in the warmed plots (a 13% increase). The results from our experiment suggest that global warming could aggravate allergic hazards and thereby jeopardize public health.

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.

Gene expression in the dimorphic sperm cells of Plumbago zeylanica: transcript profiling, diversity, and relationship to cell type.

Plumbago zeylanica produces cytoplasmically dimorphic sperm cells that target the egg and central cell during fertilization. In mature pollen, the larger sperm cell contains numerous mitochondria, is associated with the vegetative nucleus (S(vn)), and fuses preferentially with the central cell, forming endosperm. The other, plastid-enriched sperm cell (S(ua)) fuses with the egg cell, forming the zygote and embryo. Sperm expressed genes were investigated using ESTs produced from each sperm type; differential expression was validated through suppression subtractive hybridization, custom microarrays, real-time RT-PCR and in situ hybridization. The expression profiles of dimorphic sperm cells reflect a diverse and broad complement of genes, including high proportions of conserved and unknown genes, as well as distinct patterns of expression. A number of genes were highly up-regulated in the male germ line, including some genes that were differentially expressed in either the S(ua) or the S(vn). Differentially up-regulated genes in the egg-targeted S(ua) showed increased expression in transcription and translation categories, whereas the central cell-targeted S(vn) displayed expanded expression in the hormone biosynthesis category. Interestingly, the up-regulated genes expressed in the sperm cells appeared to reflect the expected post-fusion profiles of the future embryo and endosperm. As sperm cytoplasm is known to be transmitted during fertilization in this plant, sperm-contributed mRNAs are probably transported during fertilization, which could influence early embryo and endosperm development.

Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage.

Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10,732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development.

Integrated metagenomics and network analysis of soil microbial community of the forest timberline.

The forest timberline responds quickly and markedly to climate changes, rendering it a ready indicator. Climate warming has caused an upshift of the timberline worldwide. However, the impact on belowground ecosystem and biogeochemical cycles remain elusive. To understand soil microbial ecology of the timberline, we analyzed microbial communities via 16s rRNA Illumina sequencing, a microarray-based tool named GeoChip 4.0 and a random matrix theory-based association network approach. We selected 24 sampling sites at two vegetation belts forming the timberline of Shennongjia Mountain in Hubei Province of China, a region with extraordinarily rich biodiversity. We found that temperature, among all of measured environmental parameters, showed the most significant and extensive linkages with microbial biomass, microbial diversity and composition at both taxonomic and functional gene levels, and microbial association network. Therefore, temperature was the best predictor for microbial community variations in the timberline. Furthermore, abundances of nitrogen cycle and phosphorus cycle genes were concomitant with NH4+-N, NO3−-N and total phosphorus, offering tangible clues to the underlying mechanisms of soil biogeochemical cycles. As the first glimpse at both taxonomic and functional compositions of soil microbial community of the timberline, our findings have major implications for predicting consequences of future timberline upshift.

Long-term cultivation of a stable Methylocystis-dominated methanotrophic enrichment enabling tailored production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Methane (CH4) is a readily available feedstock for production of polyhydroxyalkanoates (PHAs). The structure and PHA production capacity of a Methylocystis-dominated methanotrophic enrichment was stable in long-term operation (>175 days) when grown exponentially under non-aseptic conditions in fill-and-draw batch cultures with ammonium as nitrogen source. Cells harvested in the draw step were incubated in the absence of nitrogen with various combinations of CH4 and valerate to assess capacity for synthesis of poly(3-hydroxybutyrate) (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). When fed CH4 alone, only P3HB was produced. When fed CH4 plus valerate, PHBV was synthesized. The mol% of 3-hydroxyvalerate (3HV) increased with added valerate. Oxidation of CH4 was required for valerate assimilation, and the fraction of CH4 oxidized increased with increased mol% 3 HV. By separating PHA accumulation from cell replication, tailored PHA-rich biomass can be generated by addition of co-substrate, while retaining a large inoculum for the next cycle of cell division.

Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana : behavior during transport, maturation and upon dissociation of male germ unit associations

The promoter sequence of sperm-expressed gene, PzIPT isolated from the Svn (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.