Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality.

Cereal crop by-products are a promising source of renewable raw material for the production of biofuel from lignocellulose. However, their enzymatic conversion to fermentable sugars is detrimentally affected by lignins. Here the characterization of the Brachypodium Bd5139 mutant provided with a single nucleotide mutation in the caffeic acid O-methyltransferase BdCOMT6 gene is reported. This BdCOMT6-deficient mutant displayed a moderately altered lignification in mature stems. The lignin-related BdCOMT6 gene was also found to be expressed in grains, and the alterations of Bd5139 grain lignins were found to mirror nicely those evidenced in stem lignins. The Bd5139 grains displayed similar size and composition to the control. Complementation experiments carried out by introducing the mutated gene into the AtCOMT1-deficient Arabidopsis mutant demonstrated that the mutated BdCOMT6 protein was still functional. Such a moderate down-regulation of lignin-related COMT enzyme reduced the straw recalcitrance to saccharification, without compromising the vegetative or reproductive development of the plant.

Endomembrane proteomics reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers.

Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called 'the bran' is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel).

The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing.

RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of pentatricopeptide repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the C terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue. Here, we describe two mutants, otp71 and otp72, disrupted in genes encoding mitochondrial E-type PPR proteins with single editing defects in ccmFN 2 and rpl16 transcripts, respectively. Comparisons between the E domains of these proteins and previously reported editing factors from chloroplasts suggested that there are characteristic differences in the proteins between the two organelles. To test this, we swapped E domains between two mitochondrial and two chloroplast editing factors. In all cases investigated, E domains from the same organelle (chloroplast or mitochondria) were found to be exchangeable; however, swapping the E domain between organelles led to non-functional editing factors. We conclude that the E domains of mitochondrial and plastid PPR proteins are not functionally equivalent, and therefore that an important component of the putative editing complexes in the two organelles may be different.

New Growth-Related Features of Wheat Grain Pericarp Revealed by Synchrotron-Based X-ray Micro-Tomography and 3D Reconstruction.

Wheat (Triticum aestivum L.) is one of the most important crops as it provides 20% of calories and proteins to the human population. To overcome the increasing demand in wheat grain production, there is a need for a higher grain yield, and this can be achieved in particular through an increase in the grain weight. Moreover, grain shape is an important trait regarding the milling performance. Both the final grain weight and shape would benefit from a comprehensive knowledge of the morphological and anatomical determinism of wheat grain growth. Synchrotron-based phase-contrast X-ray microtomography (X-ray µCT) was used to study the 3D anatomy of the growing wheat grain during the first developmental stages. Coupled with 3D reconstruction, this method revealed changes in the grain shape and new cellular features. The study focused on a particular tissue, the pericarp, which has been hypothesized to be involved in the control of grain development. We showed considerable spatio-temporal diversity in cell shape and orientations, and in tissue porosity associated with stomata detection. These results highlight the growth-related features rarely studied in cereal grains, which may contribute significantly to the final grain weight and shape.

OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1.

Over 20 proteins of the pentatricopeptide repeat (PPR) family have been demonstrated to be involved in RNA editing in plant mitochondria and chloroplasts. All of these editing factors contain a so-called 'E' domain that has been shown to be essential for editing to occur. The presumption has been that this domain recruits the (unknown) editing enzyme to the RNA. In this report, we show that not all putative E-class PPR proteins are directly involved in RNA editing. Disruption of the OTP70 gene leads to a strong defect in splicing of the plastid transcript rpoC1, leading to a virescent phenotype. The mutant has a chloroplast transcript pattern characteristic of a reduction in plastid-encoded RNA polymerase activity. The E domain of OTP70 is not required for splicing, and can be deleted or replaced by the E domain from the known editing factor CRR4 without loss of rpoC1 splicing. Furthermore, the E domain of OTP70 is incapable of inducing RNA editing when fused to the RNA binding domain of CRR4. We conclude that the truncated E domain of OTP70 is no longer functional in RNA editing, and that the protein has acquired a new function in promoting RNA splicing.

A study of new Arabidopsis chloroplast RNA editing mutants reveals general features of editing factors and their target sites.

RNA editing in higher plant organelles results in the conversion of specific cytidine residues to uridine residues in RNA. The recognition of a specific target C site by the editing machinery involves trans-acting factors that bind to the RNA upstream of the C to be edited. In the last few years, analysis of mutants affected in chloroplast biogenesis has identified several pentatricopeptide repeat (PPR) proteins from the PLS subfamily that are essential for the editing of particular RNA transcripts. We selected other genes from the same subfamily and used a reverse genetics approach to identify six new chloroplast editing factors in Arabidopsis thaliana (OTP80, OTP81, OTP82, OTP84, OTP85, and OTP86). These six factors account for nine editing sites not previously assigned to an editing factor and, together with the nine PPR editing proteins previously described, explain more than half of the 34 editing events in Arabidopsis chloroplasts. OTP80, OTP81, OTP85, and OTP86 target only one editing site each, OTP82 two sites, and OTP84 three sites in different transcripts. An analysis of the target sites requiring the five editing factors involved in editing of multiple sites (CRR22, CRR28, CLB19, OTP82, and OTP84) suggests that editing factors can generally distinguish pyrimidines from purines and, at some positions, must be able to recognize specific bases.

Spatial correlation of water distribution and fine structure of arabinoxylans in the developing wheat grain.

This study was to investigate the distribution of water and arabinoxylan structures in growing wheat grain using two complementary imaging techniques, magnetic resonance microimaging (µMRI) and mass spectrometry imaging (MSI). µMRI showed an inhomogeneous water distribution, particularly at early stages. This heterogeneity revealed histological differences that corresponded, within the limits of resolution of µMRI, to tissues with specific physiological functions, including the vascular bundles, the cavity and the endosperm periphery. All of these tissues had a higher water content than the central endosperm. MSI revealed distinct xylan structures in these regions with high levels of Araf substitution around the cavity and acetylated xylans concentrated at the endosperm periphery. For the first time, acetylation and Araf substitution of arabinoxylans were found by image processing to spatially correlate with water distribution in planta. Acetylation and Araf substitution of xylans, which alter chain-chain interactions and increase wall porosity, decreased as the grain matured.

The endosperm cavity of wheat grains contains a highly hydrated gel of arabinoxylan.

Cereal grains provide a substantial part of the calories for humans and animals. The main quality determinants of grains are polysaccharides (mainly starch but also dietary fibers such as arabinoxylans, mixed-linkage glucans) and proteins synthesized and accumulated during grain development in a specialized storage tissue: the endosperm. In this study, the composition of a structure localized at the interface of the vascular tissues of the maternal plant and the seed endosperm was investigated. This structure is contained in the endosperm cavity where water and nutrients are transferred to support grain filling. While studying the wheat grain development, the cavity content was found to autofluoresce under UV light excitation. Combining multispectral analysis, Fourier-Transform infrared spectroscopy, immunolabeling and laser-dissection coupled with wet chemistry, we identified in the cavity arabinoxylans and hydroxycinnamic acids. The cavity content forms a "gel" in the developing grain, which persists in dry mature grain and during subsequent imbibition. Microscopic magnetic resonance imaging revealed that the gel is highly hydrated. Our results suggest that arabinoxylans are synthesized by the nucellar epidermis, released in the cavity where they form a highly hydrated gel which might contribute to regulate grain hydration.

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of ß-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

The Arabidopsis gene YS1 encoding a DYW protein is required for editing of rpoB transcripts and the rapid development of chloroplasts during early growth.

Virescence, a phenotype in which leaves green more slowly than usual, is recognized to play a role in protection from photo-oxidative damage before healthy chloroplasts are developed. The elucidation of the molecular mechanisms underlying virescence will provide insights into how the development of chloroplasts is controlled. In this study, we find that knockout alleles of Yellow Seedlings 1 (YS1) in Arabidopsis lead to a virescent phenotype, which disappears by 3 weeks after germination. The ys1 mutation resulted in marked decreases in photosynthetic capacity and photosynthetic pigment complexes, and disturbed ultrastructure of thylakoid membranes in 8-day-old seedlings. However, cotyledons of ys1 seedlings pre-treated in the dark for 5 days turn green almost as fast as the wild type in light, revealing that the developmental defects in ys1 are limited to the first few days after germination. Inspection of all known plastid RNA editing and splicing events revealed that YS1 is absolutely required for editing of site 25992 in rpoB transcripts encoding the beta subunit of the plastid-encoded RNA polymerase (PEP). YS1 is a nuclear-encoded chloroplast-localized pentatricopeptide repeat protein differing from previously described editing factors in that it has a C-terminal DYW motif. A defect in PEP activity is consistent with the changes in plastid transcript patterns observed in ys1 seedlings. We conclude that the activity of PEP containing RpoB translated from unedited transcripts is insufficient to support rapid chloroplast differentiation.

Plant RNA editing.

In plants, post-transcriptional modification of transcripts includes C-to-U, U-to-C and A-to-I editing. RNA editing in plants is essential, with many mutants impaired in editing of specific sites exhibiting strong deleterious phenotypes, even lethality. The majority of editing in plants occurs in mitochondrial and plastid transcripts, however, A-to-I editing also occurs in cytosolic tRNAs. Here we review recent findings concerning the cellular machineries involved in the different types of editing, recent analysis of the proposed functions for editing, and recent models for its appearance and retention in different plant lineages.

CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts.

RNA editing changes the sequence of many transcripts in plant organelles, but little is known about the molecular mechanisms determining the specificity of the process. In this study, we have characterized CLB19 (also known as PDE247), a gene that is required for editing of two distinct chloroplast transcripts, rpoA and clpP. Loss-of-function clb19 mutants present a yellow phenotype with impaired chloroplast development and early seedling lethality under greenhouse conditions. Transcript patterns are profoundly affected in the mutant plants, with a pattern entirely consistent with a defect in activity of the plastid-encoded RNA polymerase. CLB19 encodes a pentatricopeptide repeat protein similar to the editing specificity factors CRR4 and CRR21, but, unlike them, is implicated in editing of two target sites.

A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons.

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana. A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health.

Use of X-ray micro computed tomography imaging to analyze the morphology of wheat grain through its development.

BACKGROUND: Wheat is one of the most important staple source in the world for human consumption, animal feed and industrial raw materials. To deal with the global and increasing population demand, enhancing crop yield by increasing the final weight of individual grain is considered as a feasible solution. Morphometric analysis of wheat grain plays an important role in tracking and understanding developmental processes by assessing potential impacts on grains properties, size and shape that are major determinants of final grain weight. X-ray micro computed tomography (µCT) is a very powerful non-invasive imaging tool that is able to acquire 3D images of an individual grain, enabling to assess the morphology of wheat grain and of its different compartments. Our objective is to quantify changes of morphology during growth stages of wheat grain from 3D µCT images. METHODS: 3D µCT images of wheat grains were acquired at various development stages ranging from 60 to 310 degree days after anthesis. We developed robust methods for the identification of outer and inner tissues within the grains, and the extraction of morphometric features using 3D µCT images. We also developed a specific workflow for the quantification of the shape of the grain crease. RESULTS: The different compartments of the grain could be semi-automatically segmented. Variations of volumes of the compartments adequately describe the different stages of grain developments. The evolution of voids within wheat grain reflects lysis of outer tissues and growth of inner tissues. The crease shape could be quantified for each grain and averaged for each stage of development, helping us understand the genesis of the grain shape. CONCLUSION: This work shows that µCT acquisitions and image processing methodologies are powerful tools to extract morphometric parameters of developing wheat grain. The results of quantitative analysis revealed remarkable features of wheat grain growth. Further work will focus on building a computational model of wheat grain growth based on real 3D imaging data.

Multi-excitation hyperspectral autofluorescence imaging for the exploration of biological samples.

Many plant tissues can be observed thanks to autofluorescence of their cell wall components. Hyperspectral autofluorescence imaging using confocal microscopy is a fast and efficient way of mapping fluorescent compounds in samples with a high spatial resolution. However a huge spectral overlap is observed between molecular species. As a consequence, a new data analysis approach is needed in order to fully exploit the potential of this spectroscopic technique and extract unbiased chemical information about complex biological samples. The objective of this work is to evaluate multi-excitation hyperspectral autofluorescence imaging to identify biological components in wheat grains during their development through their spectral profiles and corresponding contribution maps using Multivariate Curve Resolution - Alternating Least-Squares (MCR-ALS), a signal unmixing algorithm under proper constraints. For this purpose two different scenarios are used: 1) analyzing the total spectral domain of data sets using MCR-ALS under non negativity constraint in both spectral and spatial modes; 2) analyzing a reduced spectral domain of data sets using MCR-ALS under non negativity in both modes and trilinearity constraint in spectral mode. Considering the original instrumental setup and our data analysis approach, we will demonstrate that extracted contribution maps and spectral profiles of constituents can provide complementary information used to identify molecules in complex biological samples.

Challenging the putative structure of mannan in wheat (Triticum aestivum) endosperm.

In wheat endosperm, mannan, is poorly documented. Nevertheless, this hemicellulosic polysaccharide might have a determinant role in wheat grain development since, in Arabidopsis thaliana, mutants with a reduced amount of mannan show an altered seed development. In order to gain knowledge about mannan in wheat, we have determined its biochemical structure in wheat endosperm where mannose content is about 0.2% (dry weight basis). We developed a method of enzymatic fingerprinting and isolated mannan-enriched fractions to decipher its fine structure. Although it is widely accepted that the class of mannan present in grass cell walls is glucomannan, our data indicate that, in wheat endosperm, this hemicellulose is only represented by short unsubstituted chains of 1,4 linked D-mannose residues and is slightly acetylated. Our study provides information regarding the interactions of mannan with other cell wall components and help to progress towards the understanding of monocot cell wall architecture and the mannan synthesis in wheat endosperm.

Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development.

Important biological, nutritional and technological roles are attributed to cell wall polymers from cereal grains. The composition of cell walls in dry wheat grain has been well studied, however less is known about cell wall deposition and modification in the grain outer layers during grain development. In this study, the composition of cell walls in the outer layers of the wheat grain (Triticum aestivum Recital cultivar) was investigated during grain development, with a focus on cell wall phenolics. We discovered that lignification of outer layers begins earlier than previously reported and long before the grain reaches its final size. Cell wall feruloylation increased in development. However, in the late stages, the amount of ferulate releasable by mild alkaline hydrolysis was reduced as well as the yield of lignin-derived thioacidolysis monomers. These reductions indicate that new ferulate-mediated cross-linkages of cell wall polymers appeared as well as new resistant interunit bonds in lignins. The formation of these additional linkages more specifically occurred in the outer pericarp. Our results raised the possibility that stiffening of cell walls occur at late development stages in the outer pericarp and might contribute to the restriction of the grain radial growth.

Cross-competition in transgenic chloroplasts expressing single editing sites reveals shared cis elements.

RNA editing in organelles of angiosperm plants results in alteration of Cs to Us in transcripts. In most editing sites analyzed in vitro or in vivo, sequences within approximately 30 nucleotides (nt) 5' and 10 nt 3' of the edited C have been found to be required for selection of the correct C editing target and for editing efficiency, but no consensus sequences have been identified. The effect of high-level expression of two different minigenes carrying either the rpoB-2 or the ndhF-2 editing site on editing was determined for all 31 known edited Cs in two transgenic tobacco lines. The editing efficiencies of both the corresponding rpoB and ndhF editing sites in the endogenous genes' transcripts and in several other genes' transcripts were reduced in the chloroplast transgenic plants. Conserved nucleotides could be identified in the sequences immediately 5' of each overexpressed editing site and in the sites in the additional genes that experienced a competition effect, though the conserved nucleotides differ 5' of rpoB-2 and 5' of ndhF-2. Inspection of sequences surrounding chloroplast and mitochondrial editing sites reveals that they can be grouped into clusters which carry conserved nucleotides within 30 nt 5' of the C target of editing. The data are consistent with a model in which the same trans factor recognizes several chloroplast or mitochondrial editing sites, depending on the particular sequence 5' of the edited C.

Developmental co-variation of RNA editing extent of plastid editing sites exhibiting similar cis-elements.

In tobacco, 30 of 34 sites in chloroplast transcripts that undergo C-to-U RNA editing can be grouped into clusters of 2-5 sites based on sequence similarities immediately 5' to the edited C. According to a previous transgenic analysis, overexpression of transcripts representing one cluster member results in reduction in editing of all cluster members, suggesting that members of an individual cluster share a trans-factor that is present in limiting amounts. To compare leaves and roots, we quantified the editing extent at 34 sites in wild-type tobacco and at three sites in spinach and Arabidopsis. We observed that transcripts of most NADH dehydrogenase subunits are edited inefficiently in roots. With few exceptions, members of the same editing site cluster co-varied in editing extent in chloroplasts versus non-green root plastids, with members of most clusters uniformly exhibiting either a high or low editing extent in roots. The start codon of the ndhD transcript must be created by editing, but the C target is edited inefficiently in roots, and no NDH-D protein could be detected upon immunoblotting. Our data are consistent with the hypothesis that cluster-specific trans-factors exist and that some are less abundant in roots, limiting the editing extent of certain sites in root plastids.

Breeding maize for silage and biofuel production, an illustration of a step forward with the genome sequence.

The knowledge of the gene families mostly impacting cell wall digestibility variations would significantly increase the efficiency of marker-assisted selection when breeding maize and grass varieties with improved silage feeding value and/or with better straw fermentability into alcohol or methane. The maize genome sequence of the B73 inbred line was released at the end of 2009, opening up new avenues to identify the genetic determinants of quantitative traits. Colocalizations between a large set of candidate genes putatively involved in secondary cell wall assembly and QTLs for cell wall digestibility (IVNDFD) were then investigated, considering physical positions of both genes and QTLs. Based on available data from six RIL progenies, 59 QTLs corresponding to 38 non-overlapping positions were matched up with a list of 442 genes distributed all over the genome. Altogether, 176 genes colocalized with IVNDFD QTLs and most often, several candidate genes colocalized at each QTL position. Frequent QTL colocalizations were found firstly with genes encoding ZmMYB and ZmNAC transcription factors, and secondly with genes encoding zinc finger, bHLH, and xylogen regulation factors. In contrast, close colocalizations were less frequent with genes involved in monolignol biosynthesis, and found only with the C4H2, CCoAOMT5, and CCR1 genes. Close colocalizations were also infrequent with genes involved in cell wall feruloylation and cross-linkages. Altogether, investigated colocalizations between candidate genes and cell wall digestibility QTLs suggested a prevalent role of regulation factors over constitutive cell wall genes on digestibility variations.