The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis.

The most abundant and renewable source of carbohydrates is found in the lignocellulosic biomass of plants, contained in the cell walls surrounding plant cells. However, the plant cell wall has evolved to be rigid and nearly impenetrable, making the conversion of plant biomass to simple sugars a laborious and expensive endeavour. After cellulose, a class of polysaccharides called hemicelluloses make up the majority of the carbohydrate content of plant biomass. The β-(1,4)-linked xylose homopolymer xylan is the most abundant hemicellulose in secondary cell walls of angiosperms (Scheller and Ulvskov, 2010). Many genes with unique, if not well understood, roles in xylan biosynthesis have been identified. Members of two glycosyltransferase (GT) families are required for synthesis of the xylan backbone: IRREGULAR XYLEM 9 (IRX9) and IRX14 from GT43 and IRX10 from GT47 (Brown et al., 2009; Rennie and Scheller, 2014; Peña et al., 2007). In Arabidopsis thaliana, each protein has a single homolog with partial functional redundancy: IRX9L, IRX14L and IRX10L, respectively. Due to redundancy, not all the single mutants have a strong phenotype, but in general, mutants in these genes and their paralogs have reduced xylan content, shorter xylan chains and lower endogenous xylan synthase activity (Brown et al., 2009; Rennie and Scheller, 2014; Peña et al., 2007). The reduced xylan often results in thinner and weaker secondary cell walls, leading to the irregular collapsed xylem vessels for which the genes are named. Only IRX10 and IRX10L have been demonstrated to have β-(1,4)-xylosyltransferase activity in vitro (Urbanowicz et al., 2014; Jensen et al., 2014). The lack of evidence for their catalytic roles has led to the hypothesis of a xylan synthase complex wherein IRX9(L) and/or IRX14(L) play primarily structural roles and IRX10(L) is responsible for elongation of the xylan backbone. While its interaction with cellulose makes xylan a critical part of the structural integrity of the cell wall, it also makes xylan a contributor to the recalcitrance of plant biomass to enzymatic degradation. An additional downstream obstacle presented by xylan is the large amount of pentoses and acetyl groups released during its enzymatic breakdown. Pentoses cannot be efficiently metabolized by and may inhibit the fermentation of hexoses like glucose in commonly used microbial platforms, for example Saccharomyces, and acetate is generally detrimental to their growth (Young et al., 2010). Therefore, reducing the amount of xylan in the plant secondary cell wall is an important goal in the development of biofuel crops. The ideal strategy would be to reduce the amount of xylan in a wild-type plant since few knockout mutant lines of relevant biofuel crops exist. Knockout mutants could be generated using genome editing, but in many cases, multiple genes would have to be targeted to achieve the desired phenotype; for example, there are four homologs of IRX10 in sorghum and seven in rice. Here, we report the dominant suppression of xylan biosynthesis in wild-type A. thaliana via the overexpression of two mutated forms of IRX10. The amino acid sequence of AtIRX10 was aligned with those of the closest homologs in A. thaliana and a range of plant species (Oryza sativa, Populus trichocarpa, Brachypodium distachyon, Plantago ovata, Physcomitrella patens), as well as the Homo sapiens homolog Exostosin1 (EXT1) (Figure 1a). The degree of conservation of amino acid residues was used to infer potential roles of specific residues in catalysis and identify targets for mutation. One amino acid in particular, Glycine-283, is conserved in all sequences and is known to be important for the function of EXT1 in humans (Duncan et al., 2001). G283 is clustered with three other residues that are fully conserved across land plants: F277, C278 and E293. The proximity of these residues to G283 and their large and/or charged functional groups made these residues attractive additional targets. F277 and C278 were mutated to alanine, and G283 was mutated to aspartate. E293 was mutated to glutamine to maintain the size but remove the charge of the functional group. A fifth conserved residue in a different portion of the protein, H146, was mutated to aspartate. Each single amino acid substituted version of the IRX10 coding sequence, as well as the native sequence, was then used to create individual transgenic A. thaliana lines with a wild-type Col-0 background. The coding sequences were expressed with a C-terminal FLAG epitope fusion under the strong, constitutive cauliflower mosaic virus 35S promoter. The first generation of transgenic plants carrying the F277A, G283D and E293Q forms of IRX10 displayed significantly reduced height, while lines expressing the H146D and C277A mutants more closely resembled the empty vector control (EVC) (Figure 1b). Dwarfed stature is an early indication that xylan biosynthesis may be suppressed. Immunoblotting using anti-FLAG antibody served to confirm that the mutated proteins were expressed and that silencing was not taking place. Only one line (E293Q) expressed highly enough to be detectable in a crude protein extract. However, expression in all IRX10 and G283D lines was detectable once proteins from the stem were solubilized and enriched via anti-FLAG immunoprecipitation (Figure 1c). While the F277A lines displayed a strong phenotype, we could not confirm expression of the mutated IRX10. The plants were not further analysed, since the phenotype could be due to co-suppression of IRX10 with the transgene. When the monosaccharide composition of the stem was analysed, each of the mutant IRX10 overexpressors demonstrated varying degrees of xylose reduction, with the G283D and E293Q mutants being the most significant (Figure 1d). The xylose content of plants overexpressing the native form of IRX10 was comparable to EVC. The effects of the drastically reduced xylose content in G283D and E293Q T2 stems were further investigated with xylan immunolabelling and toluidine blue O staining of transverse sections of the basal stem (Figure 1e). The xylem vessels of EVC plants and the native IRX10 overexpressors are characteristically large and round in shape with thick secondary cell walls. The xylem vessels of E293Q overexpressors, in contrast, are smaller and more irregular in shape with significantly thinner secondary cell walls. The G283D overexpressors exhibit a less pronounced irregular vessel phenotype despite the thin secondary cell walls and an even greater reduction in xylose than the E293Q overexpressors. This work reveals that there are at least two amino acid residues, G283 and E293, which are critically important in the xylan biosynthesis activity of IRX10. The fact that overexpressing a mutated form of IRX10 is capable of disrupting the activity of the native IRX10 is consistent with the existence of a xylan synthase complex. The more abundant IRX10 mutant protein is apparently able to displace the functional IRX10 in the complex, thereby abrogating xylan biosynthetic activity. We do not have a method to directly determine the amount of native IRX10 and IRX10L in the plants, but xylan synthase activity assay with microsomes from stems of the transgenic plants confirmed a lower activity in the G283D and E293Q plants (Figure 1f). The proteins that IRX10 and IRX10L interact directly with in a biosynthetic complex have not been unambiguously identified, but there is significant evidence that IRX9(L) and IRX14(L) are part of the complex. It is conceivable that additional proteins are also part of the complex (Jiang et al., 2016). From a practical perspective, the findings reported here readily suggest a method to improve biomass composition. Down-regulation of xylan biosynthesis in bioenergy crops is desirable because an increased hexose/pentose ratio is advantageous for the downstream processing of biomass (Young et al., 2010). Obviously, in the practical implementation, down-regulation must be done in a way that does not cause a yield reduction. Prior work has demonstrated that when xylan is specifically reduced in interfascicular fibre cells, while maintained in vessels, the resulting plants have a large increase in hexose/pentose ratio while not showing any reduction in growth (Petersen, 2012). Hence, the expression of the mutated IRX10 forms described here under strong fibre-specific promoters would be a potential way to engineer plants with the desired properties. Alternatively, the expression of the mutated forms in vessels could be eliminated in various ways. Considering the degree of protein sequence conservation, our approach could be easily adapted to a variety of relevant crop species. The biosynthetic pathway targeted in this paper is xylan biosynthesis. However, the same kind of dominant negative approach can be used in most cases that involve protein complexes. Dominant negative protein variants could be used as a biotechnology tool in other systems to reduce biosynthetic activity, especially where genetic redundancy makes genome editing or mutagenesis impractical. This work conducted by the Joint BioEnergy Institute was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under contract no. DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. AGB was supported in part by a ARCS Foundation fellowship. AGB and HVS are inventors on a patent application related to the work described here. AGB and HVS designed this study and wrote the manuscript. DB analysed monosaccharide composition and AGB conducted all other experiments.

Lignocellulosic biomass can be used to produce biofuels, bioproducts or biopower and presents a renewable energy resource that has the capacity to lower the global carbon footprint. However, shifting climates leading to an increase in arid lands and limits on water available for irrigation make it imperative to understand how the water deficit these bioenergy crops face may impact their biomass and bioenergy characteristics. In collaboration with the EpiCon project, we are reporting the results from the first trial comparing well-watered RT430 sorghum lines with RT430 lines that have been subjected to drought. Our results indicate that while there are differences in biomass characteristics of sorghum vegetative tissue in response to post-flowering drought stress, these changes are relatively small in comparison to total sugar composition. Notable changes appear in pectic monosaccharides (rhamnose, arabinose, galactose, galacturonic acid) of newly expanding tissues, in addition to hemicellulosic changes in older tissues (glucose, mannose, glucuronic acid). Consistent with the relatively small monosaccharide changes, the saccharification efficiencies of biomass differed only little between plants with and without postflowering drought. In general, the results suggesting that post-flowering drought stress has little impact on biomass characteristics.

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall while avoiding the activation of the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene, which confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [14 C]- acetyl-CoA to oligogalacturonides. Through site-directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5, pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibility to multiple strains of the generalist necrotroph Botrytis cinerea and have decreased camalexin production upon B. cinerea infection. These results illustrate that cell wall composition is intimately connected to fungal disease resistance, and outline a potential route for engineering powdery mildew resistance into susceptible crop species. This article is protected by copyright. All rights reserved.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatran-scriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Background Switchgrass ( Panicum virgatum L.) is a promising bioenergy feedstock because it can be grown on marginal land and produces abundant biomass. Recalcitrance of the lignocellulosic components of the switchgrass cell wall to enzymatic degradation into simple sugars impedes efficient biofuel production. We previously demonstrated that overexpression of OsAT10, a BAHD acyltransferase gene, enhances saccharification efficiency in rice. Results Here we show that overexpression of the rice OsAT10 gene in switchgrass decreased the levels of cell wall-bound ferulic acid (FA) in green leaf tissues and to a lesser extent in senesced tissues, and significantly increased levels of cell wall-bound p -coumaric acid ( p -CA) in green leaves but decreased its level in senesced tissues of the T 0 plants under greenhouse conditions. The engineered switchgrass lines exhibit an approximate 40% increase in saccharification efficiency in green tissues and a 30% increase in senesced tissues. Conclusion Our study demonstrates that overexpression of OsAT10 , a rice BAHD acyltransferase gene, enhances saccharification of lignocellulosic biomass in switchgrass.

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side-chains of rhamnogalacturonan-I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing β-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-β-l-Arap to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a 10-fold higher affinity. Transfer of Arap to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

BACKGROUND:Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production.RESULTS:We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells.CONCLUSION:The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.

BACKGROUND:We previously developed several strategies to engineer plants to produce cost-efficient biofuels from plant biomass. Engineered Arabidopsis plants with low xylan and lignin content showed normal growth and improved saccharification efficiency under standard growth conditions. However, it remains to be determined whether these engineered plants perform well under drought stress, which is the primary source of abiotic stress in the field.RESULTS:Upon exposing engineered Arabidopsis plants to severe drought, we observed better survival rates in those with a low degree of xylan acetylation, low lignin, and low xylan content compared to those in wild-type plants. Increased pectic galactan content had no effect on drought tolerance. The drought-tolerant plants exhibited low water loss from leaves, and drought-responsive genes (RD29A, RD29B, DREB2A) were generally up-regulated under drought stress, which did not occur in the well-watered state. When compared with the wild type, plants with low lignin due to expression of QsuB, a 3-dehydroshikimate dehydratase, showed a stronger response to abscisic acid (ABA) in assays for seed germination and stomatal closure. The low-lignin plants also accumulated more ABA in response to drought than the wild-type plants. On the contrary, the drought tolerance in the engineered plants with low xylan content and low xylan acetylation was not associated with differences in ABA content or response compared to the wild type. Surprisingly, we found a significant increase in galactose levels and sugar released from the low xylan-engineered plants under drought stress.CONCLUSIONS:This study shows that plants engineered to accumulate less lignin or xylan are more tolerant to drought and activate drought responses faster than control plants. This is an important finding because it demonstrates that modification of secondary cell walls does not necessarily render the plants less robust in the environment, and it shows that substantial changes in biomass composition can be achieved without compromising plant resilience.

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

In plants, L-arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDP-Araf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transported back into the lumen. This step is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Thus, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgi-localized UDP-Araf transporters in Arabidopsis The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.

ELISA absorbance values for RG-I fractions from NbPAGR-silenced and virus-infected, control N. benthamiana plants as well as 35S::PAGR-YFP and wild type Arabidopsis plants probed with a diverse array of plant cell wall glycan-directed monoclonal antibodies. (XLSX 21 kb)

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth.Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1: 1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content.Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.