Leonardo D. Gomez

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579‐containing proteins that are essential for normal xylan deposition in the secondary cell wall

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.

AtTPS1-mediated trehalose 6-phosphate synthesis is essential for embryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells.

Trehalose and associated metabolites are part of the sugar signalling system in plants and have profound effects on development. Disruption of the TREHALOSE 6-PHOSPHATE SYNTHASE (TPS1) gene in Arabidopsis results in delayed embryo growth, altered cell wall morphology and carbon metabolism and abortion at the torpedo stage. Here we investigate the role of the TPS1 gene in post-embryonic development using two approaches. In the first we use the seed-specific ABI3 promoter to drive the TPS1 cDNA during embryo development, resulting in rescue of the embryo-lethal tps1 phenotype. Lack of expression from the ABI3::TPS1 transgene in post-germinative tps1 seedlings results in severe growth arrest, accumulation of soluble sugars and starch and leads to an increase in expression of genes related to ABA signalling. In the second approach we use TILLING (targeted induced local lesions in genomes) to generate three weaker, non-embryo-lethal, alleles (tps1-11, tps1-12 and tps1-13) and use these to demonstrate that the TPS1 protein plays a key role in modulating trehalose 6-phosphate (T6P) levels in vegetative tissues of Arabidopsis. All three weaker alleles give a consistent phenotype of slow growth and delayed flowering. Germination of tps1-11, tps1-12 and tps1-13 is hypersensitive to ABA with the degree of hypersensitivity correlating with the decrease in T6P levels in the different alleles. Stomatal pore aperture is regulated by ABA, and this was found to be affected in tps1-12. Our results show that the TPS1 gene product plays an essential role in regulating the growth of vegetative as well as embryogenic tissue in a mechanism involving ABA and sugar metabolism.

Expansins expression is associated with grain size dynamics in wheat (Triticum aestivum L.)

Grain weight is one of the most important components of cereal yield and quality. A clearer understanding of the physiological and molecular determinants of this complex trait would provide an insight into the potential benefits for plant breeding. In the present study, the dynamics of dry matter accumulation, water uptake, and grain size in parallel with the expression of expansins during grain growth in wheat were analysed. The stabilized water content of grains showed a strong association with final grain weight (r2=0.88, P <0.01). Grain length was found to be the trait that best correlated with final grain weight (r2=0.98, P <0.01) and volume (r2=0.94, P <0.01). The main events that defined final grain weight occurred during the first third of grain-filling when maternal tissues (the pericarp of grains) undergo considerable expansion. Eight expansin coding sequences were isolated from pericarp RNA and the temporal profiles of accumulation of these transcripts were monitored. Sequences showing high homology with TaExpA6 were notably abundant during early grain expansion and declined as maturity was reached. RNA in situ hybridization studies revealed that the transcript for TaExpA6 was principally found in the pericarp during early growth in grain development and, subsequently, in both the endosperm and pericarp. The signal in these images is likely to be the sum of the transcript levels of all three sequences with high similarity to the TaExpA6 gene. The early part of the expression profile of this putative expansin gene correlates well with the critical periods of early grain expansion, suggesting it as a possible factor in the final determination of grain size.

Automated saccharification assay for determination of digestibility in plant materials

BackgroundCell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies.ResultsWe developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared.ConclusionsThe automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Unlocking the potential of lignocellulosic biomass through plant science

The aim of producing sustainable liquid biofuels and chemicals from lignocellulosic biomass remains high on the sustainability agenda, but is challenged by the costs of producing fermentable sugars from these materials. Sugars from plant biomass can be fermented to alcohols or even alkanes, creating a liquid fuel in which carbon released on combustion is balanced by its photosynthetic capture. Large amounts of sugar are present in the woody, nonfood parts of crops and could be used for fuel production without compromising global food security. However, the sugar in woody biomass is locked up in the complex and recalcitrant lignocellulosic plant cell wall, making it difficult and expensive to extract. In this paper, we review what is known about the major polymeric components of woody plant biomass, with an emphasis on the molecular interactions that contribute to its recalcitrance to enzymatic digestion. In addition, we review the extensive research that has been carried out in order to understand and reduce lignocellulose recalcitrance and enable more cost-effective production of fuel from woody plant biomass.

The role of trehalose-6-phosphate synthase in Arabidopsis embryo development

We previously showed that trehalose-6-phosphate synthase 1 (TPS1), which catalyses the first step in trehalose synthesis, is essential for embryo maturation in Arabidopsis. The tps1 mutant embryos develop more slowly than wild type. Patterning in the tps1 embryos appears normal but they do not progress past the torpedo stage to cotyledon stage, which is when storage reserves start to accumulate in the expanding cotyledons. Our initial data led to the hypothesis that trehalose metabolism plays a key role in regulating storage reserve accumulation by allowing the embryo to respond to the dramatic increase in sucrose levels that occurs at the torpedo stage of embryo development. More recent data demonstrate that while the tps1 mutant is blocked in the developmental progression of embryos from torpedo to cotyledon stage the expression of genes involved in the accumulation of storage reserves proceeds in a similar fashion to wild type. Thus it appears that induction of metabolic processes required for accumulation of storage reserves in tps1 occurs independently of the developmental stage and instead follows a temporal programme similar to wild-type seeds in the same silique.

Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants

Significance Bioethanol produced from waste biomass from crops has the potential to provide a sustainable alternative to petroleum-based transportation fuel that does not compete with human food supply. The main obstacle to this approach is the resistance of this biomass to digestion. Thus, expensive energetic pretreatment and high enzyme inputs are needed to increase digestion. In this study, we screened a population of randomly mutated plants for digestibility with the aim of identifying novel factors that impact on this trait. We found a number of mutants with high digestibility and no impairments in growth or fitness. These mutants show a range of alterations in cell-wall composition, and we have mapped and characterized the mutant with the highest increase in digestibility. Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce crops with cell walls that are more susceptible to hydrolysis to reduce preprocessing and enzyme inputs. To deepen our understanding of the molecular genetic basis of lignocellulose recalcitrance, we have screened a mutagenized population of the model grass Brachypodium distachyon for improved saccharification with an industrial polysaccharide-degrading enzyme mixture. From an initial screen of 2,400 M2 plants, we selected 12 lines that showed heritable improvements in saccharification, mostly with no significant reduction in plant size or stem strength. Characterization of these putative mutants revealed a variety of alterations in cell-wall components. We have mapped the underlying genetic lesions responsible for increased saccharification using a deep sequencing approach, and here we report the mapping of one of the causal mutations to a narrow region in chromosome 2. The most likely candidate gene in this region encodes a GT61 glycosyltransferase, which has been implicated in arabinoxylan substitution. Our work shows that forward genetic screening provides a powerful route to identify factors that impact on lignocellulose digestibility, with implications for improving feedstock for cellulosic biofuel production.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan l. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non-cellulosic polysaccharides of embryos. Arabinose levels decline to approximately 15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.

A new perspective in bio-refining: levoglucosenone and cleaner lignin from waste biorefinery hydrolysis lignin by selective conversion of residual saccharides

An unexpected opportunity is reported to improve the sustainability of biorefineries whereby 8 wt% levoglucosenone (LGE) can be derived from unconverted saccharides in a lignin-rich biorefinery waste stream in a highly selective fashion (>90%). Additionally, in the process a purer lignin is obtained which can be used for further processing or materials applications. LGE is a valuable and versatile product with a plethora of applications.