Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

Advances in Physiological, Transcriptomic, Proteomic, Metabolomic, and Molecular Genetic Approaches for Enhancing Mango Fruit Quality.

Mango (Mangifera indica L.) is a nutritionally important fruit of high nutritive value, delicious in taste with an attractive aroma. Due to their antioxidant and therapeutic potential, mango fruits are receiving special attention in biochemical and pharmacognosy-based studies. Fruit quality determines consumer's acceptance, and hence, understanding the physiological, biochemical, and molecular basis of fruit development, maturity, ripening, and storage is essential. Transcriptomic, metabolomic, proteomic, and molecular genetic approaches have led to the identification of key genes, metabolites, protein candidates, and quantitative trait loci that are associated with enhanced mango fruit quality. The major pathways that determine the fruit quality include amino acid metabolism, plant hormone signaling, carbohydrate metabolism and transport, cell wall biosynthesis and degradation, flavonoid and anthocyanin biosynthesis, and carotenoid metabolism. Expression of the polygalacturonase, cutin synthase, pectin methyl esterase, pectate lyase, ß-galactosidase, and ethylene biosynthesis enzymes are related to mango fruit ripening, flavor, firmness, softening, and other quality processes, while genes involved in the MAPK signaling pathway, heat shock proteins, hormone signaling, and phenylpropanoid biosynthesis are associated with diseases. Metabolomics identified volatiles, organic acids, amino acids, and various other compounds that determine the characteristic flavor and aroma of the mango fruit. Molecular markers differentiate the mango cultivars based on their geographical origins. Genetic linkage maps and quantitative trait loci studies identified regions in the genome that are associated with economically important traits. The review summarizes the applications of omics techniques and their potential applications toward understanding mango fruit physiology and their usefulness in future mango breeding.

Chemical and Molecular Characterization of Wound-Induced Suberization in Poplar (Populus alba × P. tremula) Stem Bark.

Upon mechanical damage, plants produce wound responses to protect internal tissues from infections and desiccation. Suberin, a heteropolymer found on the inner face of primary cell walls, is deposited in specific tissues under normal development, enhanced under abiotic stress conditions and synthesized by any tissue upon mechanical damage. Wound-healing suberization of tree bark has been investigated at the anatomical level but very little is known about the molecular mechanisms underlying this important stress response. Here, we investigated a time course of wound-induced suberization in poplar bark. Microscopic changes showed that polyphenolics accumulate 3 days post wounding, with aliphatic suberin deposition observed 5 days post wounding. A wound periderm was formed 9 days post wounding. Chemical analyses of the suberin polyester accumulated during the wound-healing response indicated that suberin monomers increased from 0.25 to 7.98 mg/g DW for days 0 to 28, respectively. Monomer proportions varied across the wound-healing process, with an overall ratio of 2:1 (monomers:glycerol) found across the first 14 days post wounding, with this ratio increasing to 7:2 by day 28. The expression of selected candidate genes of poplar suberin metabolism was investigated using qRT-PCR. Genes queried belonging to lipid polyester and phenylpropanoid metabolism appeared to have redundant functions in native and wound-induced suberization. Our data show that, anatomically, the wounding response in poplar bark is similar to that described in periderms of other species. It also provides novel insight into this process at the chemical and molecular levels, which have not been previously studied in trees.

Heterologous over-expression of ACC SYNTHASE8 (ACS8) in Populus tremula x P. alba clone 717-1B4 results in elevated levels of ethylene and induces stem dwarfism and reduced leaf size through separate genetic pathways.

Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism.

Identification, characterization of an AP2/ERF transcription factor that promotes adventitious, lateral root formation in Populus.

Using activation tagging in Populus, we have identified five mutant lines showing changes in their adventitious rooting. Among the affected lines, three showed increased and two decreased adventitious rooting. We have positioned the tag in the mutant lines via recovering genomic sequences flanking the left-hand border of the activation tagging vector and validated the transcriptional activation of the proximal genes. We further characterized one line in which the cause of the observed rooting phenotype was up-regulation of a gene encoding a transcription factor of the AP2/ERF family of unknown function (PtaERF003). We show, through retransformation, that this gene has a positive effect on both adventitious and lateral root proliferation. Comparative expression analyses show that the phenotype does not result from ectopic expression but rather up-regulation of the native expression pattern of the gene. PtaERF003 function is linked to auxin signal transduction pathway, as suggested by the rapid induction and accentuated phenotypes of the transgenic plants in presence of the hormone. Upregulation of PtaERF003 led to most significant metabolic changes in the shoot suggesting of a broader regulatory role of the gene that is not restricted to root growth and development. Our study shows that dominant tagging approaches in poplar can successfully identify novel molecular factors controlling adventitious and lateral root formation in woody plants. Such discoveries can lead to technologies that can increase root proliferation and, thus, have significant economic and environmental benefits.

Members of the LATERAL ORGAN BOUNDARIES DOMAIN transcription factor family are involved in the regulation of secondary growth in Populus.

Regulation of secondary (woody) growth is of substantial economic and environmental interest but is poorly understood. We identified and subsequently characterized an activation-tagged poplar (Populus tremula × Populus alba) mutant with enhanced woody growth and changes in bark texture caused primarily by increased secondary phloem production. Molecular characterization of the mutation through positioning of the tag and retransformation experiments shows that the phenotype is conditioned by activation of an uncharacterized gene that encodes a novel member of the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors. Homology analysis showed highest similarity to an uncharacterized LBD1 gene from Arabidopsis thaliana, and we consequently named it Populus tremula × Populus alba (Pta) LBD1. Dominant-negative suppression of Pta LBD1 via translational fusion with the repressor SRDX domain caused decreased diameter growth and suppressed and highly irregular phloem development. In wild-type plants, LBD1 was most highly expressed in the phloem and cambial zone. Two key Class I KNOTTED1-like homeobox genes that promote meristem identity in the cambium were downregulated, while an Altered Phloem Development gene that is known to promote phloem differentiation was upregulated in the mutant. A set of four LBD genes, including the LBD1 gene, was predominantly expressed in wood-forming tissues, suggesting a broader regulatory role of these transcription factors during secondary woody growth in poplar.

Endogenous overexpression of Populus MYB186 increases trichome density, improves insect pest resistance, and impacts plant growth.

Trichomes are specialized epidermal cells that generally play a role in reducing transpiration and act as a deterrent to herbivory. In a screen of activation-tagged Populus tremula × Populus alba 717-1B4 trees, we identified a mutant line, fuzzy, with increased foliar trichome density. This mutant also had a 35% increase in growth rate and a 200% increase in the rate of photosynthesis as compared with wild-type poplar. The fuzzy mutant had significant resistance to feeding by larvae of the white-spotted tussock moth (Orgyia leucostigma), a generalist insect pest of poplar trees. The fuzzy trichome phenotype is attributable to activation tagging and increased expression of the gene encoding PtaMYB186, which is related to Arabidopsis thaliana MYB106, a known regulator of trichome initiation. The fuzzy phenotype can be recapitulated by overexpressing PtaMYB186 in poplar. PtaMYB186 overexpression results in reconfiguration of the poplar transcriptome, with changes in the transcript abundance of suites of genes that are related to trichome differentiation. It is notable that a plant with misexpression of a gene responsible for trichome development also had altered traits related to growth rate and pest resistance, suggesting that non-intuitive facets of plant development might be useful targets for plant improvement.

Ethylene receptor ETR2 controls trichome branching by regulating microtubule assembly in Arabidopsis thaliana.

The single-celled trichome of Arabidopsis thaliana is a widely used model system for studying cell development. While the pathways that control the later stages of trichome development are well characterized, the early signalling events that co-ordinate these pathways are less well understood. Hormones such as gibberellic acid, salicylic acid, cytokinins, and ethylene are known to affect trichome initiation and development. To understand the role of the plant hormone ethylene in trichome development, an Arabidopsis loss-of-function ethylene receptor mutant, etr2-3, which has completely unbranched trichomes, is analysed in this study. It was hypothesized that ETR2 might affect the assembly of the microtubule cytoskeleton based on analysis of the cytoskeleton in developing trichomes, and exposures to paclitaxol and oryzalin, which respectively act either to stabilize or depolymerize the cytoskeleton. Through epistatic and gene expression analyses it is shown that ETR2 is positioned upstream of CHROMATIN ASSEMBLY FACTOR1 and TRYPTICHON and is independent of the GLABRA2 and GLABRA3 pathways. These results help extend understanding of the early events that control trichome development and identify a signalling pathway through which ethylene affects trichome branching.

An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches.

BACKGROUND: Functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenized plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenized to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes. RESULTS: We have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T3 generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented. CONCLUSION: This publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.

Ethylene levels are regulated by a plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase.

Control of the levels of the plant hormone ethylene is crucial in the regulation of many developmental processes and stress responses. Ethylene production can be controlled by altering endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene or by altering its conversion to ethylene. ACC is known to be irreversibly broken down by bacterial or fungal ACC deaminases (ACDs). Sequence analysis revealed two putative ACD genes encoded for in the genome of Arabidopsis thaliana (A. thaliana) and we detected ACD activity in plant extracts. Expression of one of these A. thaliana genes (AtACD1) in bacteria indicated that it had ACD activity. Moreover, transgenic plants harboring antisense constructs of the gene decreased ACD activity to 70% of wild-type (WT) levels, displayed an increased sensitivity to ACC and produced significantly more ethylene. Taken together, these results show that AtACD1 can act as a regulator of ACC levels in A. thaliana.

Plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase activity implicated in different aspects of plant development.

Proper plant development is dependent on the coordination and tight control of a wide variety of different signals. In the study of the plant hormone ethylene, control of the immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is of interest as the level of ethylene can either help or hinder plant growth during times of stress. It is known that ACC can be reversibly removed from the biosynthesis pathway through conjugation into other compounds. We recently reported that plants can also irreversibly remove ACC from ethylene production through the activity of a plant encoded ACC deaminase. Heretofore only found in bacteria, we showed that there was ACC deaminase activity in both Arabidopsis and in developing wood of poplar. Here we extend this original work and show that there is also ACC deaminase activity in tomato plants, and that this activity is regulated during tomato fruit development. Further, using an antisense construct of AtACD1 in Arabidopsis, we investigate the role of ACC deamination during salt stress. Together these studies shed light on a new level of control during ethylene production in a wide variety of plant species and during different plant developmental stages.

Potato expressed sequence tag generation and analysis using standard and unique cDNA libraries.

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5' end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3' ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences.

How plants make tubes.

The plant body requires the transport of various materials over large distances. Two cell types that bear a striking resemblance morphologically are the cells specialized for water transport and those responsible for the transport of oxygen: xylem and lysigenous aerenchyma, respectively. Each of these cell types undergoes programmed cell death and cellular autolysis, resulting in the production of a functional space within the plant body. The major morphological difference observed is the presence of the lignified secondary wall in water-conducting tissues. The prevalence of tubular structures in other plant tissues suggests that the ability to form spaces through cellular autolysis is a fundamental paradigm in plant development and evolution.

Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood.

Ethylene is produced in wood-forming tissues, and when applied exogenously, it has been shown to cause profound effects on the pattern and rate of wood development. However, the molecular regulation of ethylene biosynthesis during wood formation is poorly understood. We have characterised an abundant 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene (PttACO1) in the wood-forming tissues of Populus tremula (L.) x P. tremuloides (Michx). PttACO1 is primarily expressed in developing secondary xylem, and is specifically upregulated during secondary wall formation. Nevertheless, according to GC-MS analysis combined with tangential cryosectioning, the distribution of ACC was found to be fairly uniform across the cambial-region tissues. Gravitational stimulation, which causes tension wood to form on the upper side of the stem, resulted in a strong induction of PttACO1 expression and ACC oxidase activity in the tension wood-forming tissues. The ACC levels increased in parallel to the PttACO1 expression. However, the increase on the upper (tension wood) side was only minor, whereas large amounts of both ACC and its hydrolysable conjugates accumulated on the lower (opposite) side of the stem. This suggests that the relatively low level of ACC on the tension wood side is a result of its conversion to ethylene by the highly upregulated PttACO1, and the concurrent accumulation of ACC on the opposite side of the wood is because of the low PttACO1 levels. We conclude that PttACO1 and ACC oxidase activity, but not ACC availability, are important in the control of the asymmetric ethylene production within the poplar stem when tension wood is induced by gravitational stimulation.

Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis.

The cambial K+ content of poplar increases during the growth period in a K+ supply dependent manner. Upon K+ starvation or application of tetraethylammoniumchloride (TEA+), a K+ channel blocker, the average vessel lumen and expansion zone area were significantly reduced. In search for the molecular basis of potassium-dependent xylogenesis in poplar, K+ transporters homologous to those of known function in Arabidopis phloem- and xylem-physiology were isolated from a poplar wood EST library. The expression profile of three distinct K+ channel types and one K+ transporter, Populus tremula K+ uptake transporter 1 (PtKUP1), was analysed by quantitative RT-PCR. Thereby, we found P. tremula outward rectifying K+ channel (PTORK) and P. tremula K+ channel 2 (PTK2) correlated with the seasonal wood production. K+ transporter P. tremula 1 (KPT1) was predominantly found in guard cells. Following the heterologous expression in Xenopus oocytes the biophysical properties of the different channels were determined. PTORK, upon membrane de-polarization mediates potassium release. PTK2 is almost voltage independent, carrying inward K+ flux at hyperpolarized potential and K+ release upon de-polarization. PtKUP1 was expressed in a K+ uptake-deficient Escherichia coli strain, where this K+ transporter rescued K+-dependent growth. In order to link the different K+ transporters to the cambial activity and wood production, we compared the expression profiles to seasonal changes in the K+ content of the bark as well as xylem vessel diameter. Thereby, we found PTORK and PTK2 transcripts to follow the annual K+ variations in poplar branches. PtKUP1 was expressed at a low level throughout the year, suggesting a housekeeping function. From these data, we conclude that K+ channels are involved in the regulation of K+-dependent wood production.

Secondary xylem development in Arabidopsis: a model for wood formation.

Our understanding of the molecular controls regulating the identity of the vascular cambium and the development of secondary xylem and phloem have not yet benefited much from the use of Arabidopsis as a genetic system. Under appropriate growth conditions Arabidopsis undergoes extensive secondary growth in the hypocotyl, with the development of both a vascular and a cork cambium. The secondary xylem of the hypocotyl develops in two phases, an early phase in which only vessel elements mature and a later stage in which both vessel elements and fibres are found. During this second phase the secondary xylem of Arabidopsis closely resembles the anatomy of the wood of an angiosperm tree, and can be used to address basic questions about wood formation. The development of the vascular cambium and secondary growth in Arabidopsis hypocotyl is described and its utility as a model for wood formation in trees is considered.