[Show abstract][Hide abstract] ABSTRACT: JASMONATE ZIM-domain (JAZ) proteins play important roles in plant defence and growth by regulating jasmonate signalling. Through
data mining, we discovered that the JAZ7 gene was up-regulated in darkness. In the dark, the jaz7 mutant displayed more severe leaf yellowing, quicker chlorophyll degradation, and higher hydrogen peroxide accumulation compared
with wild-type (WT) plants. The mutant phenotype of dark-induced leaf senescence could be rescued in the JAZ7-complemented and -overexpression lines. Moreover, the double mutants of jaz7 myc2 and jaz7 coi1 exhibited delayed leaf senescence. We further employed GeneChip analysis to study the molecular mechanism. Some key genes
down-regulated in the triple mutant myc2 myc3 myc4 were up-regulated in the jaz7 mutant under darkness. The Gene Ontology terms ‘leaf senescence’ and ‘cell death’ were significantly enriched in the differentially
expressed genes. Combining the genetic and transcriptomic analyses together, we proposed a model whereby darkness can induce
JAZ7, which might further block MYC2 to suppress dark-induced leaf senescence. In darkness, the mutation of JAZ7 might partially
liberate MYC2/MYC3/MYC4 from suppression, leading the MYC proteins to bind to the G-box/G-box-like motifs in the promoters,
resulting in the up-regulation of the downstream genes related to indole-glucosinolate biosynthesis, sulphate metabolism,
callose deposition, and JA-mediated signalling pathways. In summary, our genetic and transcriptomic studies established the
JAZ7 protein as an important regulator in dark-induced leaf senescence.
Full-text · Article · Nov 2015 · Journal of Experimental Botany
[Show abstract][Hide abstract] ABSTRACT: The utilization of lignin for fungible fuels and chemicals represents one of the most imminent challenges in modern biorefineries. However, bioconversion of lignin is highly challenging due to its recalcitrant nature as a phenolic heteropolymer. This study addressed the challenges by revealing the chemical and biological mechanisms for synergistic lignin degradation by a bacterial and enzymatic system, which significantly improved lignin consumption, cell growth and lipid yield. The Rhodococcus opacus cell growth increased exponentially in response to the level of laccase treatment, indicating the synergy between laccase and bacterial cells in lignin degradation. Other treatments like iron and hydrogen peroxide showed limited impact on cell growth. Chemical analysis of lignin under various treatments further confirmed the synergy between laccase and cells at the chemical level. 31P nuclear magnetic resonance (NMR) suggested that laccase, R. opacus cell and Fenton reaction reagents promoted the degradation of different types of lignin functional groups, elucidating the chemical basis for the synergistic effects. 31P NMR further revealed that laccase treatment had the most significant impact for degrading the abundant chemical groups. The results were further confirmed by the molecular weight analysis and lignin quantification by the Prussian blue assay. The cell–laccase fermentation led to a 17-fold increase of lipid production. Overall, the study indicated that laccase and R. opacus can synergize to degrade lignin efficiently, likely through rapid utilization of monomers generated by laccase to promote the reaction toward depolymerization. The study provided a potential path for more efficient lignin conversion and development of consolidated lignin conversion.
[Show abstract][Hide abstract] ABSTRACT: Understanding the molecular mechanisms for aromatic compound degradation is crucial for the development of effective bioremediation strategies. We report the discovery of a novel phenomenon for improved degradation of Direct Red 5B azo dye by Irpex lacteus CD2 with lignin as a co-substrate. Transcriptomics analysis was performed to elucidate the molecular mechanisms of aromatic degradation in white rot fungus by comparing dye, lignin, and dye/lignin combined treatments. A full spectrum of lignin degradation peroxidases, oxidases, radical producing enzymes, and other relevant components were up-regulated under DR5B and lignin treatments. Lignin induced genes complemented the DR5B induced genes to provide essential enzymes and redox conditions for aromatic compound degradation. The transcriptomics analysis was further verified by manganese peroxidase (MnP) protein over-expression, as revealed by proteomics, dye decolorization assay by purified MnP and increased hydroxyl radical levels, as indicated by an iron reducing activity assay. Overall, the molecular and genomic mechanisms indicated that effective aromatic polymer degradation requires synergistic enzymes and radical-mediated oxidative reactions to form an effective network of chemical processes. This study will help to guide the development of effective bioremediation and biomass degradation strategies.
No preview · Article · Oct 2015 · Journal of hazardous materials
[Show abstract][Hide abstract] ABSTRACT: The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.
Full-text · Article · Jun 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Photosynthetic hydrocarbon production bypasses the traditional biomass hydrolysis process and represents the most direct conversion of sunlight energy into the next-generation biofuels. As a major class of biologically derived hydrocarbons with diverse structures, terpenes are also valuable in producing a variety of fungible bioproducts in addition to the advanced ‘drop-in’ biofuels. However, it is highly challenging to achieve the efficient redirection of photosynthetic carbon and reductant into terpene biosynthesis. In this review, we discuss four major scientific and technical barriers for photosynthetic terpene production and recent advances to address these constraints. Collectively, photosynthetic terpene production needs to be optimized in a systematic fashion, in which the photosynthesis improvement, the optimization of terpene biosynthesis pathway, the improvement of key enzymes and the enhancement of sink effect through terpene storage or secretion are all important. New advances in synthetic biology also offer a suite of potential tools to design and engineer photosynthetic terpene platforms. The systemic integration of these solutions may lead to ‘disruptive’ technologies to enable biofuels and bioproducts with high efficiency, yield and infrastructure compatibility.
No preview · Article · Feb 2015 · Plant Biotechnology Journal
[Show abstract][Hide abstract] ABSTRACT: Lignin utilization during biomass conversion has been a major challenge for lignocellulosic biofuel. In particular, the conversion of lignin along with carbohydrate for fungible fuels and chemicals will both improve the overall carbon efficiency and reduce the need for chemical pretreatments. However, few biomass-converting microorganisms have the capacity to degrade all cell wall components including lignin, cellulose, and hemicellulose. We hereby evaluated a unique oleaginous fungus strain Cunninghamella echinulata FR3 for its capacity to degrade lignin during biomass conversion to lipid, and the potential to carry out consolidated fermentation without chemical pretreatment, especially when combined with sorghum (Sorghum bicolor) bmr mutants with reduced lignin content. The study clearly showed that lignin was consumed together with carbohydrate during biomass conversion for all sorghum samples, which indicates this organism has the potential for biomass conversion without chemical pretreatment. Even though dilute acid pretreatment of biomass resulted in more weight loss during fungal fermentation than untreated biomass, the lipid yields were comparable for untreated bmr6/bmr12 double mutant and dilute acid-pretreated wild-type biomass samples. The mechanisms for lignin degradation in oleaginous fungi were further elucidated through transcriptomics and chemical analysis. The studies showed that in C. echinulata FR3, Fenton Reaction may play an important role in lignin degradation. This discovery is among the first to show that a mechanism for lignin degradation similar to ones found in white and brown rot basidiomycetous fungi exists in an oleaginous fungus. This study suggests that oleaginous fungus such as C. echinulata FR3 can be employed for complete biomass utilization in a consolidated platform without chemical pretreatment, or can be used to convert lignin waste into lipids.
[Show abstract][Hide abstract] ABSTRACT: Efficient degradation and utilization of lignocellulosic biomass remains a challenge for sustainable and affordable biofuels. Various natural biomass utilization systems (NBUS) evolved the capacity to combat the recalcitrance of plant cell walls. The study of these NBUS could enable the development of efficient and cost-effective biocatalysts, microorganisms, and bioprocesses for biofuels and bioproducts. Here, we reviewed the recent research progresses for several NBUS, ranging from single cell microorganisms to consortiums such as cattle rumen and insect guts. These studies aided the discovery of biomass-degrading enzymes and the elucidation of the evolutionary and functional relevance in these systems. In particular, advances in the next generation 'omics' technologies offered new opportunities to explore NBUS in a high-throughput manner. Systems biology helped to facilitate the rapid biocatalyst discovery and detailed mechanism analysis, which could in turn guide the reverse design of engineered microorganisms and bioprocesses for cost-effective and efficient biomass conversion.
Full-text · Article · Mar 2014 · Current Opinion in Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.
Preview · Article · Oct 2013 · Nature Reviews Genetics
[Show abstract][Hide abstract] ABSTRACT: Genetically engineered (GE) ringspot virus-resistant papayas cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.
We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/mul of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.
This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.
[Show abstract][Hide abstract] ABSTRACT: High abundance proteins like Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. In order to address this challenge, we developed and evaluated Polyethyleneimine (PEI) Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery.
No preview · Article · Aug 2013 · Molecular & Cellular Proteomics
[Show abstract][Hide abstract] ABSTRACT: Metagenome analysis of the gut symbionts of three different insects was conducted as a means of comparing taxonomic and metabolic diversity of gut microbiomes to diet and life history of the insect hosts. A second goal was the discovery of novel biocatalysts for biorefinery applications. Grasshopper and cutworm gut symbionts were sequenced and compared with the previously identified metagenome of termite gut microbiota. These insect hosts represent three different insect orders and specialize on different food types. The comparative analysis revealed dramatic differences among the three insect species in the abundance and taxonomic composition of the symbiont populations present in the gut. The composition and abundance of symbionts was correlated with their previously identified capacity to degrade and utilize the different types of food consumed by their hosts. The metabolic reconstruction revealed that the gut metabolome of cutworms and grasshoppers was more enriched for genes involved in carbohydrate metabolism and transport than wood-feeding termite, whereas the termite gut metabolome was enriched for glycosyl hydrolase (GH) enzymes relevant to lignocellulosic biomass degradation. Moreover, termite gut metabolome was more enriched with nitrogen fixation genes than those of grasshopper and cutworm gut, presumably due to the termite's adaptation to the high fiber and less nutritious food types. In order to evaluate and exploit the insect symbionts for biotechnology applications, we cloned and further characterized four biomass-degrading enzymes including one endoglucanase and one xylanase from both the grasshopper and cutworm gut symbionts. The results indicated that the grasshopper symbiont enzymes were generally more efficient in biomass degradation than the homologous enzymes from cutworm symbionts. Together, these results demonstrated a correlation between the composition and putative metabolic functionality of the gut microbiome and host diet, and suggested that this relationship could be exploited for the discovery of symbionts and biocatalysts useful for biorefinery applications.
[Show abstract][Hide abstract] ABSTRACT: Comparison of grasshopper (G) and cutworm (C) gut microbiome with termite (T) gut microbiome showed the enrichment of energy production and conversion COGs.
[Show abstract][Hide abstract] ABSTRACT: The effect of temperature and pH conditions on enzyme activities (mean ± SD) for the four enzymes cloned from cutworm and grasshopper microbiomes. A and B. One endoglucanases from grasshopper (GH-EG1) and one from cutworm (CW-EG1) gut microbiomes. C and D. one xylanase from grasshopper (GH-Xyn1) and one from cutworm (CW-Xyn1) gut microbiomes.
[Show abstract][Hide abstract] ABSTRACT: Phylogenetic tree of 16S rRNA annotated sequences. A. Grasshopper; B, cutworm. A total of 96 and 53 partial 16S rRNA sequences were extracted from cutworm and grasshopper gut microbiomes, respectively. The sequences were then aligned with the NAST aligner, and imported into an ARB database. The nearest aligned full length sequences were used for classification and phylogenetic tree construction using RAxML. Genbank accession numbers were presented in the figure.