Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

Medicago truncatula PHO2 genes have distinct roles in phosphorus homeostasis and symbiotic nitrogen fixation.

Three PHO2-like genes encoding putative ubiquitin-conjugating E2 enzymes of Medicago truncatula were characterized for potential roles in phosphorous (P) homeostasis and symbiotic nitrogen fixation (SNF). All three genes, MtPHO2A, B and C, contain miR399-binding sites characteristic of PHO2 genes in other plant species. Distinct spatiotemporal expression patterns and responsiveness of gene expression to P- and N-deprivation in roots and shoots indicated potential roles, especially for MtPHO2B, in P and N homeostasis. Phenotypic analysis of pho2 mutants revealed that MtPHO2B is integral to Pi homeostasis, affecting Pi allocation during plant growth under nutrient-replete conditions, while MtPHO2C had a limited role in controlling Pi homeostasis. Genetic analysis also revealed a connection between Pi allocation, plant growth and SNF performance. Under N-limited, SNF conditions, Pi allocation to different organs was dependent on MtPHO2B and, to a lesser extent, MtPHO2C and MtPHO2A. MtPHO2A also affected Pi homeostasis associated with nodule formation. Thus, MtPHO2 genes play roles in systemic and localized, i.e., nodule, P homeostasis affecting SNF.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

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.

Spectroscopic analysis reveals that soil phosphorus availability and plant allocation strategies impact feedstock quality of nutrient-limited switchgrass.

The perennial native switchgrass adapts better than other plant species do to marginal soils with low plant-available nutrients, including those with low phosphorus (P) content. Switchgrass roots and their associated microorganisms can alter the pools of available P throughout the whole soil profile making predictions of P availability in situ challenging. Plant P homeostasis makes monitoring of P limitation via measurements of plant P content alone difficult to interpret. To address these challenges, we developed a machine-learning model trained with high accuracy using the leaf tissue chemical profile, rather than P content. By applying this learned model in field trials across two sites with contrasting extractable soil P, we observed that actual plant available P in soil was more similar than expected, suggesting that adaptations occurred to alleviate the apparent P constraint. These adaptations come at a metabolic cost to the plant that have consequences for feedstock chemical components and quality. We observed that other biochemical signatures of P limitation, such as decreased cellulose-to-lignin ratios, were apparent, indicating re-allocation of carbon resources may have contributed to increased P acquisition. Plant P allocation strategies also differed across sites, and these differences were correlated with the subsequent year's biomass yields.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Three Common Symbiotic ABC Subfamily B Transporters in Medicago truncatula Are Regulated by a NIN-Independent Branch of the Symbiosis Signaling Pathway.

Several ATP-binding cassette (ABC) transporters involved in the arbuscular mycorrhizal symbiosis and nodulation have been identified. We describe three previously unreported ABC subfamily B transporters, named AMN1, AMN2, and AMN3 (ABCB for mycorrhization and nodulation), that are expressed early during infection by rhizobia and arbuscular mycorrhizal fungi. These ABCB transporters are strongly expressed in symbiotically infected tissues, including in root-hair cells with rhizobial infection threads and arbusculated cells. During nodulation, the expression of these genes is highly induced by rhizobia and purified Nod factors and is dependent on DMI3 but is not dependent on other known major regulators of infection, such as NIN, NSP1, or NSP2. During mycorrhization their expression is dependent on DMI3 and RAM1 but not on NSP1 and NSP2. Therefore, they may be commonly regulated through a distinct branch of the common symbiotic pathway. Mutants with exonic Tnt1-transposon insertions were isolated for all three genes. None of the single or double mutants showed any differences in colonization by either rhizobia or mycorrhizal fungi, but the triple amn1 amn2 amn3 mutant showed an increase in nodule number. Further studies are needed to identify potential substrates of these transporters and understand their roles in these beneficial symbioses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

PHO1 family members transport phosphate from infected nodule cells to bacteroids in Medicago truncatula.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

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/.

Application of Synthetic Peptide CEP1 Increases Nutrient Uptake Rates Along Plant Roots.

The root system of a plant provides vital functions including resource uptake, storage, and anchorage in soil. The uptake of macro-nutrients like nitrogen (N), phosphorus (P), potassium (K), and sulphur (S) from the soil is critical for plant growth and development. Small signaling peptide (SSP) hormones are best known as potent regulators of plant growth and development with a few also known to have specialized roles in macronutrient utilization. Here we describe a high throughput phenotyping platform for testing SSP effects on root uptake of multiple nutrients. The SSP, CEP1 (C-TERMINALLY ENCODED PEPTIDE) enhanced nitrate uptake rate per unit root length in Medicago truncatula plants deprived of N in the high-affinity transport range. Single structural variants of M. truncatula and Arabidopsis thaliana specific CEP1 peptides, MtCEP1D1:hyp4,11 and AtCEP1:hyp4,11, enhanced uptake not only of nitrate, but also phosphate and sulfate in both model plant species. Transcriptome analysis of Medicago roots treated with different MtCEP1 encoded peptide domains revealed that hundreds of genes respond to these peptides, including several nitrate transporters and a sulfate transporter that may mediate the uptake of these macronutrients downstream of CEP1 signaling. Likewise, several putative signaling pathway genes including LEUCINE-RICH REPEAT RECPTOR-LIKE KINASES and Myb domain containing transcription factors, were induced in roots by CEP1 treatment. Thus, a scalable method has been developed for screening synthetic peptides of potential use in agriculture, with CEP1 shown to be one such peptide.

Dissection of physiological, transcriptional, and metabolic traits in two tall fescue genotypes with contrasting drought tolerance.

Tall fescue (Festuca arundinacea) is an important cool-season perennial forage grass that forms mutualistic symbioses with fungal endophytes. Physiological, biochemical and transcriptional comparisons were made between two tall fescue genotypes with contrasting drought tolerance (tolerant, T400, and sensitive, S279), either with or without endophyte (Epichloë coenophiala). Drought stress was applied by withholding watering until plants reached mild, moderate and severe stresses. Physiological characterization showed that T400 had narrower, thicker leaves, and lower leaf conductance under well-watered conditions, compared to S279. After severe drought and recovery, endophytic T400 had greater shoot and root biomass than other plant types. Under drought, leaf osmotic pressure increased much more in T400 than S279, consistent with accumulation of metabolites/osmolytes, especially proline. Gene Ontology enrichment analysis indicated that T400 had more active organic acid metabolism than S279 under drought, and implicated the role of endophyte in stimulating protein metabolism in both genotypes. Overall T400 and S279 responded to endophyte differently in aspects of physiology, gene transcription and metabolites, indicating plant genotype-specific reactions to endophyte infection.

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation.

Knowing how switchgrass (Panicum virgatum L.) responds and adapts to phosphorus (P)-limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P-stress on genome transcription and whole-plant metabolism, physiology and development in switchgrass. P-limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P-stress, and decreased root diameter with increased density and length of root hairs at severe P-stress. RNA-seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P-stress in roots and/or shoots compared to P-replete plants, with many stress-induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P-acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P-stress in shoots and roots, although this trend reversed under severe P-stress, especially in shoots.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215-351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.