Transcriptional Programs and Regulators Underlying Age-Dependent and Dark-Induced Senescence in Medicago truncatula.

In forage crops, age-dependent and stress-induced senescence reduces forage yield and quality. Therefore, delaying leaf senescence may be a way to improve forage yield and quality as well as plant resilience to stresses. Here, we used RNA-sequencing to determine the molecular bases of age-dependent and dark-induced leaf senescence in Medicago truncatula. We identified 6845 differentially expressed genes (DEGs) in M3 leaves associated with age-dependent leaf senescence. An even larger number (14219) of DEGs were associated with dark-induced senescence. Upregulated genes identified during age-dependent and dark-induced senescence were over-represented in oxidation-reduction processes and amino acid, carboxylic acid and chlorophyll catabolic processes. Dark-specific upregulated genes also over-represented autophagy, senescence and cell death. Mitochondrial functions were strongly inhibited by dark-treatment while these remained active during age-dependent senescence. Additionally, 391 DE transcription factors (TFs) belonging to various TF families were identified, including a core set of 74 TFs during age-dependent senescence while 759 DE TFs including a core set of 338 TFs were identified during dark-induced senescence. The heterologous expression of several senescence-induced TFs belonging to NAC, WKRY, bZIP, MYB and HD-zip TF families promoted senescence in tobacco leaves. This study revealed the dynamics of transcriptomic responses to age- and dark-induced senescence in M. truncatula and identified senescence-associated TFs that are attractive targets for future work to control senescence in forage legumes.

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.).

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Increased Ascorbate Biosynthesis Does Not Improve Nitrogen Fixation Nor Alleviate the Effect of Drought Stress in Nodulated Medicago truncatula Plants.

Legume plants are able to establish nitrogen-fixing symbiotic relations with Rhizobium bacteria. This symbiosis is, however, affected by a number of abiotic constraints, particularly drought. One of the consequences of drought stress is the overproduction of reactive oxygen (ROS) and nitrogen species (RNS), leading to cellular damage and, ultimately, cell death. Ascorbic acid (AsA), also known as vitamin C, is one of the antioxidant compounds that plants synthesize to counteract this oxidative damage. One promising strategy for the improvement of plant growth and symbiotic performance under drought stress is the overproduction of AsA via the overexpression of enzymes in the Smirnoff-Wheeler biosynthesis pathway. In the current work, we generated Medicago truncatula plants with increased AsA biosynthesis by overexpressing MtVTC2, a gene coding for GDP-L-galactose phosphorylase. We characterized the growth and physiological responses of symbiotic plants both under well-watered conditions and during a progressive water deficit. Results show that increased AsA availability did not provide an advantage in terms of plant growth or symbiotic performance either under well-watered conditions or in response to drought.

Draft Genome Sequences of Switchgrass Diazotrophs.

We report the draft genome sequences of five native nitrogen-fixing bacteria associated with roots of switchgrass isolated from the tallgrass prairies of Oklahoma. Nitrogen-fixing genes, including the nif cluster, are conserved across the Klebsiella and Kosakonia strains.

The Development of Bacteriophage Resistance in Vibrio alginolyticus Depends on a Complex Metabolic Adaptation Strategy.

Lytic bacteriophages have been well documented to play a pivotal role in microbial ecology due to their complex interactions with bacterial species, especially in aquatic habitats. Although the use of phages as antimicrobial agents, known as phage therapy, in the aquatic environment has been increasing, recent research has revealed drawbacks due to the development of phage-resistant strains among Gram-negative species. Acquired phage resistance in marine Vibrios has been proven to be a very complicated process utilizing biochemical, metabolic, and molecular adaptation strategies. The results of our multi-omics approach, incorporating transcriptome and metabolome analyses of Vibrio alginolyticus phage-resistant strains, corroborate this prospect. Our results provide insights into phage-tolerant strains diminishing the expression of phage receptors ompF, lamB, and btuB. The same pattern was observed for genes encoding natural nutrient channels, such as rbsA, ptsG, tryP, livH, lysE, and hisp, meaning that the cell needs to readjust its biochemistry to achieve phage resistance. The results showed reprogramming of bacterial metabolism by transcript regulations in key-metabolic pathways, such as the tricarboxylic acid cycle (TCA) and lysine biosynthesis, as well as the content of intracellular metabolites belonging to processes that could also significantly affect the cell physiology. Finally, SNP analysis in resistant strains revealed no evidence of amino acid alterations in the studied putative bacterial phage receptors, but several SNPs were detected in genes involved in transcriptional regulation. This phenomenon appears to be a phage-specific, fine-tuned metabolic engineering, imposed by the different phage genera the bacteria have interacted with, updating the role of lytic phages in microbial marine ecology.

Improved node culture methods for rapid vegetative propagation of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). RESULTS: Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. CONCLUSIONS: In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Distinguishing HapMap Accessions Through Recursive Set Partitioning in Hierarchical Decision Trees.

The HapMap (haplotype map) projects have produced valuable genetic resources in life science research communities, allowing researchers to investigate sequence variations and conduct genome-wide association study (GWAS) analyses. A typical HapMap project may require sequencing hundreds, even thousands, of individual lines or accessions within a species. Due to limitations in current sequencing technology, the genotype values for some accessions cannot be clearly called. Additionally, allelic heterozygosity can be very high in some lines, causing genetic and sometimes phenotypic segregation in their descendants. Genetic and phenotypic segregation degrades the original accession's specificity and makes it difficult to distinguish one accession from another. Therefore, it is vitally important to determine and validate HapMap accessions before one conducts a GWAS analysis. However, to the best of our knowledge, there are no prior methodologies or tools that can readily distinguish or validate multiple accessions in a HapMap population. We devised a bioinformatics approach to distinguish multiple HapMap accessions using only a minimum number of genetic markers. First, we assign each candidate marker with a distinguishing score (DS), which measures its capability in distinguishing accessions. The DS score prioritizes those markers with higher percentages of homozygous genotypes (allele combinations), as they can be stably passed on to offspring. Next, we apply the "set-partitioning" concept to select optimal markers by recursively partitioning accession sets. Subsequently, we build a hierarchical decision tree in which a specific path represents the selected markers and the homogenous genotypes that can be used to distinguish one accession from others in the HapMap population. Based on these algorithms, we developed a web tool named MAD-HiDTree (Multiple Accession Distinguishment-Hierarchical Decision Tree), designed to analyze a user-input genotype matrix and construct a hierarchical decision tree. Using genetic marker data extracted from the Medicago truncatula HapMap population, we successfully constructed hierarchical decision trees by which the original 262 M. truncatula accessions could be efficiently distinguished. PCR experiments verified our proposed method, confirming that MAD-HiDTree can be used for the identification of a specific accession. MAD-HiDTree was developed in C/C++ in Linux. Both the source code and test data are publicly available at https://bioinfo.noble.org/MAD-HiDTree/.

Silencing Folylpolyglutamate Synthetase1 (FPGS1) in Switchgrass (Panicum virgatum L.) Improves Lignocellulosic Biofuel Production.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

GmVTL1a is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation.

Legumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for rhizobial enzyme nitrogenase that catalyses nitrogen fixation, but the SM iron transporter has not been identified. We use yeast complementation, real-time PCR and proteomics to study putative soybean (Glycine max) iron transporters GmVTL1a and GmVTL1b and have characterized the role of GmVTL1a using complementation in plant mutants, hairy root transformation and microscopy. GmVTL1a and GmVTL1b are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), which is essential for nitrogen fixation. GmVTL1a expression is enhanced in nodule infected cells and both proteins are localized to the SM. GmVTL1a transports iron in yeast and restores nitrogen fixation when expressed in the Ljsen1 mutant. Three GmVTL1a amino acid substitutions that block nitrogen fixation in Ljsen1 plants reduce iron transport in yeast. We conclude GmVTL1a is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the nitrogen-fixing symbiosis.

MtSSPdb: The Medicago truncatula Small Secreted Peptide Database.

A growing number of small secreted peptides (SSPs) in plants are recognized as important regulatory molecules with roles in processes such as growth, development, reproduction, stress tolerance, and pathogen defense. Recent discoveries further implicate SSPs in regulating root nodule development, which is of particular significance for legumes. SSP-coding genes are frequently overlooked, because genome annotation pipelines generally ignore small open reading frames, which are those most likely to encode SSPs. Also, SSP-coding small open reading frames are often expressed at low levels or only under specific conditions, and thus are underrepresented in non-tissue-targeted or non-condition-optimized RNA-sequencing projects. We previously identified 4,439 SSP-encoding genes in the model legume Medicago truncatula To support systematic characterization and annotation of these putative SSP-encoding genes, we developed the M. truncatula Small Secreted Peptide Database (MtSSPdb; https://mtsspdb.noble.org/). MtSSPdb currently hosts (1) a compendium of M. truncatula SSP candidates with putative function and family annotations; (2) a large-scale M. truncatula RNA-sequencing-based gene expression atlas integrated with various analytical tools, including differential expression, coexpression, and pathway enrichment analyses; (3) an online plant SSP prediction tool capable of analyzing protein sequences at the genome scale using the same protocol as for the identification of SSP genes; and (4) information about a library of synthetic peptides and root and nodule phenotyping data from synthetic peptide screens in planta. These datasets and analytical tools make MtSSPdb a unique and valuable resource for the plant research community. MtSSPdb also has the potential to become the most complete database of SSPs in plants.

Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation.

Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, the control of microbial infection of plant cells, the control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, the control of bacteroid differentiation, metabolism and transport supporting symbiosis, and the control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also briefly consider the future of SNF genetics in the era of pan-genomics and genome editing.

A protein complex required for polar growth of rhizobial infection threads.

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.

The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis.

Symbiotic nitrogen fixation by rhizobia in legume root nodules is a key source of nitrogen for sustainable agriculture. Genetic approaches have revealed important roles for only a few of the thousands of plant genes expressed during nodule development and symbiotic nitrogen fixation. Previously, we isolated >100 nodulation and nitrogen fixation mutants from a population of Tnt1-insertion mutants of Medigaco truncatula Using Tnt1 as a tag to identify genetic lesions in these mutants, we discovered that insertions in a M. truncatula nodule-specific polycystin-1, lipoxygenase, α-toxin (PLAT) domain-encoding gene, MtNPD1, resulted in development of ineffective nodules. Early stages of nodule development and colonization by the nitrogen-fixing bacterium Sinorhizobium meliloti appeared to be normal in the npd1 mutant. However, npd1 nodules ceased to grow after a few days, resulting in abnormally small, ineffective nodules. Rhizobia that colonized developing npd1 nodules did not differentiate completely into nitrogen-fixing bacteroids and quickly degraded. MtNPD1 expression was low in roots but increased significantly in developing nodules 4 d postinoculation, and expression accompanied invading rhizobia in the nodule infection zone and into the distal nitrogen fixation zone. A functional MtNPD1:GFP fusion protein localized in the space surrounding symbiosomes in infected cells. When ectopically expressed in tobacco (Nicotiana tabacum) leaves, MtNPD1 colocalized with vacuoles and the endoplasmic reticulum. MtNPD1 belongs to a cluster of five nodule-specific single PLAT domain-encoding genes, with apparent nonredundant functions.

Genome-wide analysis of flanking sequences reveals that Tnt1 insertion is positively correlated with gene methylation in Medicago truncatula.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

GWASpro: a high-performance genome-wide association analysis server.

SUMMARY: We present GWASpro, a high-performance web server for the analyses of large-scale genome-wide association studies (GWAS). GWASpro was developed to provide data analyses for large-scale molecular genetic data, coupled with complex replicated experimental designs such as found in plant science investigations and to overcome the steep learning curves of existing GWAS software tools. GWASpro supports building complex design matrices, by which complex experimental designs that may include replications, treatments, locations and times, can be accounted for in the linear mixed model. GWASpro is optimized to handle GWAS data that may consist of up to 10 million markers and 10 000 samples from replicable lines or hybrids. GWASpro provides an interface that significantly reduces the learning curve for new GWAS investigators. AVAILABILITY AND IMPLEMENTATION: GWASpro is freely available at https://bioinfo.noble.org/GWASPRO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Functional Analysis of Cellulose Synthase CesA4 and CesA6 Genes in Switchgrass (Panicum virgatum) by Overexpression and RNAi-Mediated Gene Silencing.

Switchgrass (Panicum virgatum L.) is a leading lignocellulosic bioenergy feedstock. Cellulose is a major component of the plant cell walls and the primary substrate for saccharification. Accessibility of cellulose to enzymatic breakdown into fermentable sugars is limited by the presence of lignin in the plant cell wall. In this study, putatively novel switchgrass secondary cell wall cellulose synthase PvCesA4 and primary cell wall PvCesA6 genes were identified and their functional role in cellulose synthesis and cell wall composition was examined by overexpression and knockdown of the individual genes in switchgrass. The endogenous expression of PvCesA4 and PvCesA6 genes varied among including roots, leaves, stem, and reproductive tissues. Increasing or decreasing PvCesA4 and PvCesA6 expression to extreme levels in the transgenic lines resulted in decreased biomass production. PvCesA6-overexpressing lines had reduced lignin content and syringyl/guaiacyl lignin monomer ratio accompanied by increased sugar release efficiency, suggesting an impact of PvCesA6 expression levels on lignin biosynthesis. Cellulose content and cellulose crystallinity were decreased, while xylan content was increased in PvCesA4 and PvCesA6 overexpression or knockdown lines. The increase in xylan content suggests that the amount of non-cellulosic cell wall polysaccharide was modified in these plants. Taken together, the results show that the manipulation of the cellulose synthase genes alters the cell wall composition and availability of cellulose as a bioprocessing substrate.

MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula.

Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Medicago truncatula copper transporter 1 (MtCOPT1) delivers copper for symbiotic nitrogen fixation.

Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper (Cu) transporter would introduce it into the cell to synthesize cupro-proteins. COPT family members in the model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M. truncatula COPT family member. Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a Cu-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a Cu-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids. These data are consistent with a model in which MtCOPT1 transports Cu from the apoplast into nodule cells to provide Cu for essential metabolic processes associated with symbiotic nitrogen fixation.

An Iron-Activated Citrate Transporter, MtMATE67, Is Required for Symbiotic Nitrogen Fixation.

Iron (Fe) is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases, and other Fe proteins in both organisms. Fe solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an Fe-activated manner. Loss of MtMATE67 gene function resulted in accumulation of Fe in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an Fe signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.