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The leaflets of the atrioventricular heart valves (AHVs) regulate the one-directional flow of blood through a coordination of the extracellular matrix components, including the collagen fibers, elastin, and glycosaminoglycans. Dysfunction of the AHVs, such as those caused by unfavorable microstructural remodeling, lead to valvular heart diseases and improper blood flow, which can ultimately cause heart failure. In order to better understand the mechanics and remodeling of the AHV leaflets and how therapeutics can inadvertently cause adverse microstructural changes, a systematic characterization of the role of each constituent in the biomechanical properties is appropriate. Previous studies have quantified the contributions of the individual microstructural components to tissue-level behavior for the semilunar valve cusps, but not for the AHV leaflets. In this study, for the first time, we quantify the relationships between microstructure and mechanics of the AHV leaflet using a three-step experimental procedure: (i) biaxial tension and stress relaxation testing of control (untreated) porcine AHV anterior leaflet specimens; (ii) enzyme treatment to remove a portion of either the collagen or elastin constituent; and (iii) biaxial tensile and stress relaxation testing of the constituent-removed (treated) specimens. We have observed that the removal of ∼100% elastin resulted in a ∼10% decrease in the tissue extensibility with biaxial tension and a ∼10% increase in the overall stress reduction with stress relaxation. In contrast, removal of 46% of the collagen content insignificantly affected tissue extensibility with biaxial tension and significantly increased stress decay (10%) with stress relaxation. These findings provide an insight into the microstructure-mechanics relationship of the AHVs and will be beneficial for future developments and refinements of microstructurally informed constitutive models for the simulation of diseased and surgically intervened AHV function. STATEMENT OF SIGNIFICANCE: This study presents, for the first time, a thorough mechanical characterization of the atrioventricular heart valve leaflets before and after enzymatic removal of elastin and collagen. We found that the biaxial tensile properties of elastin-deficient tissues and collagen-deficient are stiffer. The fact of elastin supporting low-stress valve function and collagen as the main load-bearing component was evident in a decrease in the low-tension modulus for elastin-deficient tissues and in the high-tension modulus for collagen-deficient tissues. Our quantification and experimental technique could be useful in predicting the disease-related changes in heart valve mechanics. The information obtained from this work is valuable for refining the constitutive models that describe the essential microstructure-mechanics relationship., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors of this paper have no financial or personal relationships with other people or organizations that could inappropriately influence (bias) our work., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Solid-phase microextraction (SPME) was coupled to gas chromatography mass spectrometry (GC-MS) and a method optimized to quantitatively and qualitatively measure a large array of volatile metabolites in alfalfa glandular trichomes isolated from stems, trichome-free stems, and leaves as part of a non-targeted metabolomics approach. Major SPME extraction parameters optimized included SPME fiber composition, extraction temperature, and extraction time. The optimized SPME method provided the most chemically diverse coverage of alfalfa volatile and semi-volatile metabolites using a DVB/CAR/PDMS fiber, extraction temperature of 60 °C, and an extraction time of 20 min. Alfalfa SPME-GC-MS profiles were processed using automated peak deconvolution and identification (AMDIS) and quantitative data extraction software (MET-IDEA). A total of 87 trichome, 59 stem, and 99 leaf volatile metabolites were detected after background subtraction which removed contaminants present in ambient air and associated with the fibers and NaOH/EDTA buffer solution containing CaCl 2 . Thirty-seven volatile metabolites were detected in all samples, while 15 volatile metabolites were uniquely detected only in glandular trichomes, 9 only in stems, and 33 specifically in leaves as tissue specific volatile metabolites. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of glandular trichomes, stems, and leaves showed that the volatile metabolic profiles obtained from the optimized SPME-GC-MS method clearly differentiated the three tissues (glandular trichomes, stems, and leaves), and the biochemical basis for this differentiation is discussed. Although optimized using plant tissues, the method can be applied to other types of samples including fruits and other foods.

Because of the large amount of energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system, and the developmental coordination of these organs under conditions of reduced nitrogen (N) availability remains elusive. We show that the Medicago truncatula COMPACT ROOT ARCHITECTURE2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low N. C-TERMINALLY ENCODED PEPTIDE (MtCEP1) peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of YUCCA2 ( MtYUC2 ) auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by overexpressing MtYUC2 , and conversely, a treatment with YUC inhibitors or an MtYUC2 knockout rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser 643 and Ser 924 , preventing its cleavage and thereby repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement with this interaction, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions., (© 2020 American Society of Plant Biologists. All rights reserved.)

Drought stress seriously affects crop yield, and the mechanism underlying plant resistance to drought stress via macroautophagy/autophagy is not clear. Here, we show that a dehydrin, Medicago truncatula MtCAS31 (cold acclimation-specific 31), a positive regulator of drought response, plays a key role in autophagic degradation. A GFP cleavage assay and treatment with an autophagy-specific inhibitor indicated that MtCAS31 participates in the autophagic degradation pathway and that overexpressing MtCAS31 promotes autophagy under drought stress. Furthermore, we discovered that MtCAS31 interacts with the autophagy-related protein ATG8a in the AIM-like motif YXXXI, supporting its function in autophagic degradation. In addition, we identified a cargo protein of MtCAS31, the aquaporin MtPIP2;7, by screening an M. truncatula cDNA library. We found that MtPIP2;7 functions as a negative regulator of drought response. Under drought stress, MtCAS31 facilitated the autophagic degradation of MtPIP2;7 and reduced root hydraulic conductivity, thus reducing water loss and improving drought tolerance. Taken together, our results reveal a novel function of dehydrins in promoting the autophagic degradation of proteins, which extends our knowledge of the function of dehydrins. Abbreviations: AIM: ATG8-interacting motif; ATG: autophagy-related; ATI1: ATG8-interacting protein1; BiFC: Biomolecular fluorescence complementation; CAS31: cold acclimation-specific 31; ConcA: concanamycin A; DSK2: dominant suppressor of KAR2; ER: endoplasmic reticulum; ERAD: ER-associated degradation; NBR1: next to BRCA1 gene 1; PM: plasma membrane; PIPs: plasma membrane intrinsic proteins; TALEN: transcription activator-like effector nuclease; TSPO: tryptophan-rich sensory protein/translocator; UPR: unfolded protein response; VC: vector control.

Legume-rhizobia symbiosis is a time-limited process due to the onset of senescence, which results in the degradation of host plant cells and symbiosomes. A number of transcription factors, proteases, and functional genes have been associated with nodule senescence; however, whether other proteases or transcription factors are involved in nodule senescence remains poorly understood. In this study, we identified an early nodule senescence mutant in Medicago truncatula , denoted basic helix-loop-helix transcription factor2 ( bhlh2 ), that exhibits decreased nitrogenase activity, acceleration of plant programmed cell death (PCD), and accumulation of reactive oxygen species (ROS). The results suggest that MtbHLH2 plays a negative role in nodule senescence. Nodules of wild-type and bhlh2 -TALEN mutant plants at 28 d postinoculation were used for transcriptome sequencing. The transcriptome data analysis identified a papain-like Cys protease gene, denoted MtCP77 , that could serve as a potential target of MtbHLH2. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis demonstrated that MtbHLH2 directly binds to the promoter of MtCP77 to inhibit its expression. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation. In addition, the expression of MtbHLH2 in the nodules gradually decreased from the meristematic zone to the nitrogen fixation zone, whereas the expression of MtCP77 showed enhancement. These results indicate that MtbHLH2 and MtCP77 have opposite functions in the regulation of nodule senescence. These results reveal significant roles for MtbHLH2 and MtCP77 in plant PCD, ROS accumulation, and nodule senescence, and improve our understanding of the regulation of the nodule senescence process., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula , although it remains unclear whether this is a cause or an effect of the growth impairment., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Nanocomposites consisting of polydimethylsiloxane (PDMS) and well-dispersed carbon nanotubes (CNT) can be cured by microwave radiation within a minute, forming a conductive network within the cured materials. Microwave irradiation delivers energy directly to the inner core of the nanocomposites by heating CNTs and initiating rapid polymerization of the elastomer. In this paper, nanocomposites were fabricated with CNT loadings between 0.5 wt.%–2.5 wt.% via microwave irradiation. Key properties of the nanocomposites including electrical conductivity, microstructures, CNT distribution, density, and surface effects were all characterized. The properties of microwave-cured nanocomposites were compared with those manufactured by the thermal method using a conventional oven. The microwave-curing method substantially increased the electrical conductivity of the nanocomposites due to the improved nanoparticle dispersion and likely CNT alignment. Optimal microwave-curing parameters were identified to further improve the conductivity of the nanocomposites with lowest CNT loading. A conductivity enhancement of 142.8% over thermally cured nanocomposites was achieved for nanocomposites with 1 wt.% CNTs cured via one-step microwave irradiation.

Recent studies on E3 of endoplasmic reticulum (ER)-associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant-specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER-membrane-localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss-of-function mutants displayed impaired induction of the ER stress-responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD-associated ubiquitin-conjugating enzyme MtUBC32 and Sec61-translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant-specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress., (© 2019 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

In almost all symbiotic interactions between rhizobia and leguminous plants, host flavonoid-induced synthesis of Nod factors in rhizobia is required to initiate symbiotic response in plants. In this study, we found that Lotus japonicus Nod factor receptor 5 (LjNFR5) might directly regulate flavonoid biosynthesis during symbiotic interaction with rhizobia. A yeast two-hybrid analysis revealed that a dihydroflavonol-4-reductase-like protein (LjDFL1) interacts with LjNFR5. The interaction between MtDFL1 and MtNFP, two Medicago truncatula proteins with homology to LjDFL1 and LjNFR5, respectively, was also shown, suggesting that interaction between these two proteins might be conserved in different legumes. LjDFL1 was highly expressed in root hairs and epidermal cells of root tips. Lotus ljdfl1 mutants and Medicago mtdfl1 mutants produced significantly fewer infection threads (ITs) than the wild-type control plants following rhizobial treatment. Furthermore, the roots of stable transgenic L. japonicus plants overexpressing LjDFL1 formed more ITs than control roots after exposure to rhizobia. These data indicated that LjDFL1 is a positive regulator of symbiotic signaling. However, the expression of LjDFL1 was suppressed by rhizobial treatment, suggesting that a negative feedback loop might be involved in regulation of the symbiotic response in L. japonicus.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION ( NIN ) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis -regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation., (© 2019 American Society of Plant Biologists. All rights reserved.)

Only a few RNA viruses have been discovered from archaeological samples, the oldest dating from about 750 years ago. Using ancient maize cobs from Antelope house, Arizona, dating from ca. 1,000 CE, we discovered a novel plant virus with a double-stranded RNA genome. The virus is a member of the family Chrysoviridae that infect plants and fungi in a persistent manner. The extracted double-stranded RNA from 312 maize cobs was converted to cDNA, and sequences were determined using an Illumina HiSeq 2000. Assembled contigs from many samples showed similarity to Anthurium mosaic-associated virus and Persea americana chrysovirus, putative species in the Chrysovirus genus, and nearly complete genomes were found in three ancient maize samples. We named this new virus Zea mays chrysovirus 1. Using specific primers, we were able to recover sequences of a closely related virus from modern maize and obtained the nearly complete sequences of the three genomic RNAs. Comparing the nucleotide sequences of the three genomic RNAs of the modern and ancient viruses showed 98, 96.7, and 97.4% identities, respectively. Hence, in 1,000 years of maize cultivation, this virus has undergone about 3% divergence. IMPORTANCE A virus related to plant chrysoviruses was found in numerous ancient samples of maize, with nearly complete genomes in three samples. The age of the ancient samples (i.e., about 1,000 years old) was confirmed by carbon dating. Chrysoviruses are persistent plant viruses. They infect their hosts from generation to generation by transmission through seeds and can remain in their hosts for very long time periods. When modern corn samples were analyzed, a closely related chrysovirus was found with only about 3% divergence from the ancient sequences. This virus represents the oldest known plant virus., (Copyright © 2018 American Society for Microbiology.)

The development of dental adhesive resins with long-lasting antibacterial properties is a possible solution to overcome the problem of secondary caries in modern adhesive dentistry., Objectives: (i) Synthesis and characterization of nitrogen-doped titanium dioxide nanoparticles (N_TiO 2 ), (ii) topographical, compositional and wettability characterization of thin-films (unaltered and experimental) and, (iii) antibacterial efficacy of N_TiO 2 -containing dental adhesives against Streptococcus mutans biofilms., Materials and Methods: Nanoparticles were synthesized and characterized using different techniques. Specimens (diameter = 12 mm, thickness ≅ 15 μm) of OptiBond Solo Plus (Kerr Corp., USA) and experimental adhesives [50, 67 and 80% (v/v)] were fabricated, photopolymerized (1000 mW/cm 2 , 1 min) and UV-sterilized (254 nm, 800,000 μJ/cm 2 ) for microscopy, spectroscopy, wettability and antibacterial testing. Wettability was assessed with a contact angle goniometer by dispensing water droplets (2 μL) onto four random locations of each specimen (16 drops/group). Drop profiles were recorded (1 min, 25 frames/s, 37 °C) and contact angles were calculated at time = 0 s (θ INITIAL ) and time = 59 s (θ FINAL ). Antibacterial testing was performed by growing S. mutans (UA159-ldh, JM10) biofilms for either 3 or 24 h (anaerobic conditions, 37 °C) with or without continuous light irradiation (410 ± 10 nm, 3 h = 38.75 J/cm 2 , 24 h = 310.07 J/cm 2 ) against the surfaces of sterile specimens., Results: N_TiO 2 was successfully prepared using solvothermal methods. Doped-nanoparticles displayed higher light absorption levels when compared to undoped titania. Experimental adhesives demonstrated superior antibacterial efficacy in dark conditions., Conclusions: The findings presented herein suggest that N_TiO 2 is a feasible antibacterial agent against cariogenic biofilms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Plants have evolved excellent ability of flexibly regulating the growth of organs to adapt to changing environment, for example, the modulation of lateral root development in response to environmental stresses. Despite of fundamental discovery that some microRNAs are involved in this process, the molecular mechanisms of how these microRNAs work together are still largely unknown., Results: Here we show that miR390 induced by auxin promotes lateral root growth in rice. However, this promotion can be suppressed by miR393, which is induced by various stresses and ABA (Abscisic Acid). Results that miR393 responded to ABA stronger and earlier than other stresses implied that ABA likely is authentic factor for inducing miR393. The transgenic lines respectively over-expressing miR393 and OsTAS3a (Oryza sativa Trans-Acting Short RNA precursor 3a) displayed opposite phenotypes in lateral root growth. MiR390 was found to be dominantly expressed at lateral root primordia and roots tips while miR393 mainly expressed in the base part of roots at very low level. When miR393 was up-regulated by various stresses, miR390 expression level fell down. However, the risen expression level of miR390 induced by auxin didn't affect the expression of miR393 and its target OsTIR1 (Transport Inhibitor Response 1). Together with analysis of the two transgenic lines, we provide a model of how the growth of lateral roots in rice is regulated distinctively by the 2 microRNAs., Conclusion: We propose that miR390 induced by auxin triggers the lateral root growth under normal growth conditions, meanwhile miR393 just lurks as a potentially regulative role; Once plants suffer from stresses, miR393 will be induced to negatively regulate miR390-mediated growth of lateral roots in rice.

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. Medicago truncatula NODULE ROOT1 ( MtNOOT1 ), Pisum sativum COCHLEATA1 ( PsCOCH1 ), and Lotus japonicus NOOT-BOP-COCH-LIKE1 ( LjNBCL1 ) are orthologs of Arabidopsis ( Arabidopsis thaliana ) AtBLADE-ON-PETIOLE1/2 and are members of the NBCL gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of MtNOOT1 , PsCOCH1 , and LjNBCL1 triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in M. truncatula , MtNOOT2 MtNOOT2 is the paralog of MtNOOT1 and belongs to a second legume-specific NBCL subclade, the NBCL2 clade. MtNOOT2 expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. Mtnoot2 mutants did not present any particular symbiotic phenotype; however, the loss of function of both MtNOOT1 and MtNOOT2 resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. Mtnoot1 noot2 double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules., (© 2018 American Society of Plant Biologists. All rights reserved.)

Symbiotic nitrogen fixation (SNF) in legume root nodules injects millions of tons of nitrogen into agricultural lands and provides ammonia to non-legume crops under N-deficient conditions. During plant growth and development, environmental stresses, such as drought, salt, cold, and heat stress are unavoidable. This raises an interesting question as to how the legumes cope with the environmental stress along with SNF. Under drought stress, dehydrin proteins are accumulated, which function as protein protector and osmotic substances. In this study, we found that the dehydrin MtCAS31 (cold-acclimation-specific 31) functions in SNF in Medicago truncatula during drought stress. We found that MtCAS31 is expressed in nodules and interacts with leghemoglobin MtLb120-1. The interaction between the two proteins protects MtLb120-1 from denaturation under thermal stress in vivo . Compared to wild type, cas31 mutants display a lower nitrogenase activity, a lower ATP/ADP ratio, higher expression of nodule senescence genes and higher accumulation of amyloplasts under dehydration conditions. The results suggested that MtCAS31 protects MtLb120-1 from the damage of drought stress. We identified a new function for dehydrins in SNF under drought stress, which enriches the understanding of the molecular mechanism of dehydrins.

Like Arabidopsis thaliana , the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C ( FLC ) and CONSTANS ( CO ), being absent or not having a role in flowering time function in Medicago . In Arabidopsis , the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( AtSOC1 ), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized ( MtSOC1a-MtSOC1c ). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

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., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Legume plants form symbiotic associations with either nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi, which are regulated by a set of common symbiotic signaling pathway genes. Central to the signaling pathway is the activation of the DMI3/IPD3 protein complex by Ca 2+ oscillations, and the initiation of nodule organogenesis and mycorrhizal symbiosis. DMI3 is essential for rhizobial infection and nodule organogenesis; however, ipd3 mutants have been shown to be impaired only in infection thread formation but not in root nodule organogenesis in Medicago truncatula . We identified an IPD3 -like ( IPD3L ) gene in the M. truncatula genome. A single ipd3l mutant exhibits a normal root nodule phenotype. The ipd3l/ipd3-2 double mutant is completely unable to initiate infection threads and nodule primordia. IPD3L can functionally replace IPD3 when expressed under the control of the IPD3 promoter, indicating functional redundancy between these two transcriptional regulators. We constructed a version of IPD3 that was phosphomimetic with respect to two conserved serine residues (IPD3-2D). This was sufficient to trigger root nodule organogenesis, but the increased multisite phosphorylation of IPD3 (IPD3-8D) led to low transcriptional activity, suggesting that the phosphorylation levels of IPD3 fine-tune its transcriptional activity in the root nodule symbiosis. Intriguingly, the phosphomimetic version of IPD3 triggers spontaneous root-like nodules on the roots of dmi3-1 and dmi2-1 ( DMI2 is an LRR-containing receptor-like kinase gene which is required for Ca 2+ spiking), but not on the roots of wild-type or ipd3l ipd3-2 plants. In addition, fully developed arbuscules were formed in the ipd3l ipd3-2 mutants but not the ccamk/dmi3-1 mutants. Collectively, our data indicate that, in addition to IPD3 and IPD3L , another new genetic component or other new phosphorylation sites of IPD3 function downstream of DMI3 in rhizobial and mycorrhizal symbioses.

Introduction: Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification., Objectives: Pathway-specific labeling using isotopic molecular oxygen ( 18 O 2 ) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18 O 2 -metabolome analysis., Methods: 18 O 2 labeled whole Medicago truncatula seedlings were prepared using 18 O 2 -air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC-solid-phase extraction (SPE)., Results: A total of 511 peaks labeled by 18 O 2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4'- O -[2'- O -coumaroyl-glucuronopyranosyl-(1-2)- O -glucuronopyranoside], that was labeled by 18 O 2 . To the best of our knowledge, this is the first report of apigenin 4'-glucuronide in M. truncatula . Using MS n analysis, we estimated that 18 O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18 O atom was incorporated in the 4'-hydroxy group. Thus, non-specific incorporation of an 18 O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction., Conclusion: Our finding indicated that 18 O 2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity., Competing Interests: Compliance with ethical standardsAll authors declare that they have no conflict of interest.This article does not contain any studies with human participants or animals performed by any of the authors.

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%-56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotiana tabacum (Tnt1) insertional mutant line were carried out in the one M. truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: Switchgrass is a crop with many desirable traits for bioenergy production. Plant genomes have high DNA methylation levels throughout genes and transposable elements and DNA methylation is known to play a role in silencing transposable elements. Here we analyzed methylomes in two switchgrass genotypes AP13 and VS16. AP13 is derived from a lowland ecotype and VS16, typically considered drought-tolerant, is derived from an upland ecotype, both genotypes are tetraploid (2n = 4× = 36)., Results: Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and bisulfite-sequencing (BS-seq) were used to profile DNA methylation in genomic features of AP13 and VS16. The methylation patterns in genes and transposable elements were similar to other plants, however, overall CHH methylation levels were comparatively low. Differentially methylated regions (DMRs) were assessed and a total of 1777 CG-DMRs, 573 CHG-DMRs, and 3 CHH-DMRs were detected between the two genotypes. TEs and their flanking regions were higher than that of genic regions. Different types of TEs had different methylation patterns, but the two LTRs (Copia and Gypsy) were similarly methylated, while LINEs and DNA transposons typically had different methylation patterns. MeDIP-seq data was compared to BS-seq data and most of the peaks generated by MeDIP-seq were confirmed to be highly methylated by BS-seq., Conclusions: DNA methylation in switchgrass genotypes obtained from the two ecotypes were found similar. Collinear gene pairs in two subgenomes (A and B) were not significantly differentially methylated. Both BS-seq and MeDIP-seq methodologies were found effective. Methylation levels were highest at CG and least in CHH. Increased DNA methylation was seen in TEs compared to genic regions. Exploitation of TE methylations can be a viable option in future crop improvement.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression. Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4. Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Heritable microbes are abundant in nature and influential to their hosts and the communities in which they reside. However, drivers of variability in the prevalence of heritable symbionts and their rates of transmission are poorly resolved, particularly across host populations experiencing variable biotic and abiotic environments. To fill these gaps, we surveyed 25 populations of two native grasses (Elymus virginicus and Elymus canadensis) across the southern Great Plains (USA). Both grass species host heritable endophytic fungi (genus Epichloё) and can hybridize where their ranges overlap. From a subset of hosts, we characterized endophyte genotype using genetic loci that link to bioactive alkaloid production. First, we found mean vertical transmission rates and population-level prevalence were positively correlated, specifically for E. virginicus. However, both endophyte prevalence and transmission varied substantially across populations and did not strongly correlate with abiotic variables, with one exception: endophyte prevalence decreased as drought stress decreased for E. virginicus hosts. Second, we evaluated the potential influence of biotic factors and found that, after accounting for climate, endophyte genotype explained significant variation in symbiont inheritance. We also contrasted populations where host species co-occurred in sympatry vs. allopatry. Sympatry could potentially increase interspecific hybridization, but this variable did not associate with patterns of symbiont prevalence or transmission success. Our results reveal substantial variability in symbiont prevalence and transmission across host populations and identify symbiont genotype, and to a lesser extent, the abiotic environment as sources of this variation.

Plant-herbivore interactions are often mediated by plant microorganisms, and the "defensive mutualism" of epichloid fungal endophytes of grasses is an example. These endophytes synthesize bioactive alkaloids that generally have detrimental effects on the performance of insect herbivores, but the underlying mechanisms are not well understood. Our objective was to determine whether changes in the physiology and/or behavior of aphids explain the changes in performance of insects feeding on endophytic plants. We studied the interaction between the aphid Rhopalosiphum padi and the annual ryegrass Lolium multiflorum symbiotic (E+) or not symbiotic (E-) with the fungus Epichloë occultans that can synthesize loline alkaloids. We hypothesized that aphids feeding on E+ plants have higher energetic demands for detoxification of fungal alkaloids, thereby negatively impacting the individual performance, population growth, and structure. Aphids growing on E+ plants had lower values in morphometric and functional variables of individual performance, displayed lower birth rate, smaller population size, and dramatic structural changes. However, aphids exhibited lower values of standard metabolic rate (SMR) on E+ plants, which suggests no high costs of detoxification. Behavioral variables during the first 8 h of feeding showed that aphids did not change the phloem sap ingestion with the presence of fungal endophytes. We hypothesize that aphids may maintain phloem sap ingestion according to their fungal alkaloid tolerance capacity. In other words, when alkaloid concentrations overcome tolerance threshold, ingestion of phloem should decrease, which may explain the observed lower values of SMR in E+ feeding aphids.

The soybean gene GmFWL1 (FW2-2-like1) belongs to a plant-specific family that includes the tomato FW2-2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2-2-like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain-associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process., (© 2017 John Wiley & Sons Ltd.)

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 ( TAS3 ) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/ TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 ( NSP1 ) and NSP2 Accordingly, inactivation of the miR390/ TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7 , enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/ TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a "vanillin synthase" could enable biotechnological production of vanillin from ferulic acid using a "natural" vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Plant resistance mechanisms to insect herbivory can potentially be bred into crops as an important strategy for integrated pest management. Medicago truncatula ecotypes inoculated with the rhizobium Ensifer medicae (Sinorhizobium medica) WSM419 were screened for resistance to herbivory by caterpillars of the beet armyworm, Spodoptera exigua, through leaf and whole plant choice studies; TN1.11 and F83005.5 are identified as the least and most deterrent ecotypes, respectively. In response to caterpillar herbivory, both ecotypes mount a robust burst of plant defensive jasmonate phytohormones. Restriction of caterpillars to either of these ecotypes does not adversely affect pest performance. This argues for an antixenosis (deterrence) resistance mechanism associated with the F83005.5 ecotype. Unbiased metabolomic profiling identified strong ecotype-specific differences in metabolite profile, particularly in the content of oleanolic-derived saponins that may act as antifeedants. Compared to the more susceptible ecotype, F83005.5 has higher levels of oleanolic-type zanhic acid- and medicagenic acid-derived compounds. Together, these data support saponin-mediated deterrence as a resistance mechanism of the F83005.5 ecotype and implicates these compounds as potential antifeedants that could be used in agricultural sustainable pest management strategies.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play a vital role in the response to drought stress. Here, we report a lipid-anchored NACsa TF in Medicago falcata MfNACsa is an essential regulator of plant tolerance to drought stress, resulting in the differential expression of genes involved in oxidation reduction and lipid transport and localization. MfNACsa is associated with membranes under unstressed conditions and, more specifically, is targeted to the plasma membrane through S -palmitoylation. However, a Cys 26 -to-Ser mutation or inhibition of S -palmitoylation results in MfNACsa retention in the endoplasmic reticulum/Golgi. Under drought stress, MfNACsa translocates to the nucleus through de- S -palmitoylation mediated by the thioesterase MtAPT1, as coexpression of APT1 results in the nuclear translocation of MfNACsa, whereas mutation of the catalytic site of APT1 results in colocalization with MfNACsa and membrane retention of MfNACsa. Specifically, the nuclear MfNACsa binds the glyoxalase I ( MtGlyl ) promoter under drought stress, resulting in drought tolerance by maintaining the glutathione pool in a reduced state, and the process is dependent on the APT1-NACsa regulatory module. Our findings reveal a novel mechanism for the nuclear translocation of an S -palmitoylated NAC in response to stress., (© 2017 American Society of Plant Biologists. All rights reserved.)

In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Switchgrass ( Panicum virgatum ) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O -methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT -deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

Current agricultural practices rely on heavy use of fertilizers for increased crop productivity. However, the problems associated with heavy fertilizer use, such as high cost and environmental pollution, require the development of crop species with increased nutrient use efficiency. In this study, by using transgenic approaches, we have revealed the critical role of OsNLA1 in phosphate (Pi) accumulation of rice plants. When grown under sufficient Pi and nitrate levels, OsNLA1 knockdown ( Osnla1-1, Osnla1-2 , and Osnla1-3 ) lines accumulated higher Pi content in their shoot tissues compared to wild-type, whereas, over-expression lines (OsNLA1-OE1, OsNLA1-OE2, and OsNLA1-OE3) accumulated the least levels of Pi. However, under high Pi levels, knockdown lines accumulated much higher Pi content compared to wild-type and exhibited Pi toxicity symptoms in the leaves. In contrast, the over-expression lines had 50-60% of the Pi content of wild-type and did not show such symptoms. When grown under limiting nitrate levels, OsNLA1 transgenic lines also displayed a similar pattern in Pi accumulation and Pi toxicity symptoms compared to wild-type suggesting an existence of cross-talk between nitrogen (N) and phosphorous (P), which is regulated by OsNLA1. The greater Pi accumulation in knockdown lines was a result of enhanced Pi uptake/permeability of roots compared to the wild-type. The cross-talk between N and P was found to be nitrate specific since the knockdown lines failed to over-accumulate Pi under low (sub-optimal) ammonium level. Moreover, OsNLA1 was also found to interact with OsPHO2, a known regulator of Pi homeostasis, in a Yeast Two-Hybrid (Y2H) assay. Taken together, these results show that OsNLA1 is involved in Pi homeostasis regulating Pi uptake and accumulation in rice plants and may provide an opportunity to enhance P use efficiency by manipulating nitrate supply in the soil.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: A major challenge of high throughput transcriptome studies is presenting the data to researchers in an interpretable format. In many cases, the outputs of such studies are gene lists which are then examined for enriched biological concepts. One approach to help the researcher interpret large gene datasets is to associate genes and informative terms (iTerm) that are obtained from the biomedical literature using the eGIFT text-mining system. However, examining large lists of iTerm and gene pairs is a daunting task., Results: We have developed WebGIVI, an interactive web-based visualization tool ( http://raven.anr.udel.edu/webgivi/ ) to explore gene:iTerm pairs. WebGIVI was built via Cytoscape and Data Driven Document JavaScript libraries and can be used to relate genes to iTerms and then visualize gene and iTerm pairs. WebGIVI can accept a gene list that is used to retrieve the gene symbols and corresponding iTerm list. This list can be submitted to visualize the gene iTerm pairs using two distinct methods: a Concept Map or a Cytoscape Network Map. In addition, WebGIVI also supports uploading and visualization of any two-column tab separated data., Conclusions: WebGIVI provides an interactive and integrated network graph of gene and iTerms that allows filtering, sorting, and grouping, which can aid biologists in developing hypothesis based on the input gene lists. In addition, WebGIVI can visualize hundreds of nodes and generate a high-resolution image that is important for most of research publications. The source code can be freely downloaded at https://github.com/sunliang3361/WebGIVI . The WebGIVI tutorial is available at http://raven.anr.udel.edu/webgivi/tutorial.php .

Venturia effusa is the most important pathogen of pecan in the southeastern United States. Little information exists on the population biology and genetic diversity of the pathogen. A hierarchical sampling of 784 isolates from 63 trees in 11 pecan orchards in the southeastern United States were screened against a set of 30 previously characterized microsatellite markers. Populations were collected from Georgia (n = 2), Florida (n = 1), Alabama (n = 2), Mississippi (n = 1), Louisiana (n = 1), Illinois (n = 1), Oklahoma (n = 1), Texas (n = 1), and Kansas (n = 1). Clonality was low in all orchard populations (≤10.1% of isolates), and there were consistently high levels of genotypic diversity (Shannon-Weiner's index = 3.49 to 4.59) and gene diversity (Nei's measure = 0.513 to 0.713). Analysis of molecular variance showed that, although 81% of genetic diversity occurred at the scale of the individual tree, 16% occurred between orchards and only 3% between trees within orchards. All populations could be differentiated from each other (P = 0.01), and various cluster analyses indicated that some populations were more closely related compared with other pairs of populations. This is indicative of some limited population differentiation in V. effusa in the southeastern United States. Bayesian and nearest-neighbor methods suggested eight clusters, with orchards from Georgia and Florida being grouped together. A minimum spanning tree of all 784 isolates also indicated some isolate identification with source population. Linkage disequilibrium was detected in all but one population (Kansas), although 8 of the 11 populations had <20% of loci at disequilibrium. A Mantel test demonstrated a relationship between physical and genetic distance between populations (Z = 11.9, r = 0.559, P = 0.001). None of the populations were at mutation-drift equilibrium. All but 3 of the 11 populations had a deficiency of gene diversity compared with that expected at mutation-drift equilibrium (indicating population expansion); the remaining populations had an excess of gene diversity compared with that expected at mutation-drift equilibrium (indicating a recent bottleneck). These observations are consistent with the known history of pecan and pecan scab, which is that V. effusa became an issue on cultivated pecan in the last approximately 120 years (recent population expansion). Recently reported mating type genes and the sexual stage of this fungus may help explain the observed population characteristics, which bear a strong resemblance to those of other well-characterized sexually reproducing ascomycete pathogens.

Most legume plants can form nodules, specialized lateral organs that form on roots, and house nitrogen-fixing bacteria collectively called rhizobia. The uptake of the phytohormone auxin into cells is known to be crucial for development of lateral roots. To test the role of auxin influx in nodulation we used the auxin influx inhibitors 1-naphthoxyacetic acid (1-NOA) and 2-NOA, which we found reduced nodulation of Medicago truncatula. This suggested the possible involvement of the AUX/LAX family of auxin influx transporters in nodulation. Gene expression studies identified MtLAX2 , a paralogue of Arabidopsis ( Arabidopsis thaliana ) AUX1 , as being induced at early stages of nodule development. MtLAX2 is expressed in nodule primordia, the vasculature of developing nodules, and at the apex of mature nodules. The MtLAX2 promoter contains several auxin response elements, and treatment with indole-acetic acid strongly induces MtLAX2 expression in roots. mtlax2 mutants displayed root phenotypes similar to Arabidopsis aux1 mutants, including altered root gravitropism, fewer lateral roots, shorter root hairs, and auxin resistance. In addition, the activity of the synthetic DR5-GUS auxin reporter was strongly reduced in mtlax2 roots. Following inoculation with rhizobia, mtlax2 roots developed fewer nodules, had decreased DR5-GUS activity associated with infection sites, and had decreased expression of the early auxin responsive gene ARF16a Our data indicate that MtLAX2 is a functional analog of Arabidopsis AUX1 and is required for the accumulation of auxin during nodule formation in tissues underlying sites of rhizobial infection., (© 2017 The author(s). All Rights Reserved.)

Arabidopsis thaliana Stress Associated Protein 9 (AtSAP9) is a member of the A20/AN1 zinc finger protein family known to play important roles in plant stress responses and in the mammalian immune response. Although SAPs of several plant species were shown to be involved in abiotic stress responses, the underlying molecular mechanisms are largely unknown, and little is known about the involvement of SAPs in plant disease responses. Expression of SAP9 in Arabidopsis is up-regulated in response to dehydration, cold, salinity and abscisic acid (ABA), as well as pathogen infection. Constitutive expression of AtSAP9 in Arabidopsis leads to increased sensitivity to ABA and osmotic stress during germination and post-germinative development. Plants that overexpress AtSAP9 also showed increased susceptibility to infection by non-host pathogen Pseudomonas syringae pv. phaseolicola, indicating a potential role of AtSAP9 in disease resistance. AtSAP9 was found to interact with RADIATION SENSITIVE23d (Rad23d), a shuttle factor for the transport of ubiquitinated substrates to the proteasome, and it is co-localized with Rad23d in the nucleus. Thus, AtSAP9 may promote the protein degradation process by mediating the interaction of ubiquitinated targets with Rad23d. Taken together, these results indicate that AtSAP9 regulates abiotic and biotic stress responses, possibly via the ubiquitination/proteasome pathway., (© 2016 John Wiley & Sons Ltd.)

The multidrug and toxin extrusion (MATE) transporter family comprises 70 members in the Medicago truncatula genome, and they play seemingly important, yet mostly uncharacterized, physiological functions. Here, we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export, Al 3+ tolerance and Fe translocation. MtMATE69 is a citric acid transporter induced by Fe-deficiency. Overexpression of MtMATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions. MtMATE66 is a plasma membrane citric acid transporter primarily expressed in root epidermal cells. The mtmate66 mutant had less root growth than the wild type under Al 3+ stress, and seedlings were chlorotic under Fe-deficient conditions. Overexpression of MtMATE66 rendered hairy roots more tolerant to Al 3+ toxicity. MtMATE55 is involved in seedling development and iron homeostasis, as well as hormone signaling. The mtmate55 mutant had delayed development and chlorotic leaves in mature plants. Both knock-out and overexpression mutants of MtMATE55 showed altered Fe accumulation and abnormal hormone levels compared with the wild type. We demonstrate that the zinc-finger transcription factor MtSTOP is essentially required for MtMATE66 expression and plant resistance to H + and Al 3+ toxicity. The proper expression of two previously characterized MATE flavonoid transporters MtMATE1 and MtMATE2 also depends on several transcription factors. This study reveals not only functional diversity of MATE transporters and regulatory mechanisms in legumes against H + and Al 3+ stresses, but also casts light on their role in metal nutrition and hormone signaling under various stresses., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

2,4-Dichlorophenoxyacetic acid (2,4-D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole-3-acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4-D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4-D-specific mutants suggested that 2,4-D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4-D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4-D but not IAA altered the actin structure in long-term and short-term assays. Analysis of the 2,4-D-specific mutant aar1-1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4-D-induced depolymerization of actin. The ubiquitin proteasome mutants tir1-1 and axr1-12, which show enhanced resistance to 2,4-D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4-D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4-D on the organization of actin filaments. Roots of the double mutant aar1-1 tir1-1 also showed enhanced resistance to 2,4-D-induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4-D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF TIR 1 ubiquitin proteasome components., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. Fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH have been used for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, in part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent V max of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Collectively, our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants.

Pseudomonas syringae pv. tomato strain DC3000 ( Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana . Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis ., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

Background: The native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acid O -methyltransferase ( COMT ) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years., Results: Field-grown COMT down-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35-84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25-32% increase in enzymatic sugar release after hydrothermal pretreatment. The COMT -silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7-8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weight-average molecular weights of hemicellulose were 7-11% lower in the transgenic than in the control lines. In addition, we found that silencing of COMT in switchgrass led to 20-22% increased cellulose accessibility as measured by the Simons' stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons., Conclusions: The down-regulation of COMT in switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of the COMT -silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification.

Flavonols are one of the largest groups of flavonoids that confer benefits for the health of plants and animals. Flavonol glycosides are the predominant flavonoids present in the model legume Lotus japonicus. The molecular mechanisms underlying the biosynthesis of flavonol glycosides as yet remain unknown in L. japonicus. In the present study, we identified a total of 188 UDP-glycosyltransferases (UGTs) in L. japonicus by genome-wide searching. Notably, 12 UGTs from the UGT72 family were distributed widely among L. japonicus chromosomes, expressed in all tissues, and showed different docking scores in an in silico bioinformatics docking analysis. Further enzymatic assays showed that five recombinant UGTs (UGT72AD1, UGT72AF1, UGT72AH1, UGT72V3, and UGT72Z2) exhibit activity toward flavonol, flavone, and isoflavone aglycones. In particular, UGT72AD1, UGT72AH1, and UGT72Z2 are flavonol-specific UGTs with different kinetic properties. In addition, the overexpression of UGT72AD1 and UGT72Z2 led to increased accumulation of flavonol rhamnosides in L. japonicus and Arabidopsis thaliana. Moreover, the increase of kaempferol 3-O-rhamnoside-7-O-rhamnoside in transgenic A. thaliana inhibited root growth as compared with the wild-type control. These results highlight the significance of the UGT72 family in flavonol glycosylation and the role of flavonol rhamnosides in plant growth.

Key Message: Transcriptomes of two switchgrass genotypes representing the upland and lowland ecotypes will be key tools in switchgrass genome annotation and biotic and abiotic stress functional genomics. Switchgrass (Panicum virgatum L.) is an important bioenergy feedstock for cellulosic ethanol production. We report genome-wide transcriptome profiling of two contrasting tetraploid switchgrass genotypes, VS16 and AP13, representing the upland and lowland ecotypes, respectively. A total of 268 million Illumina short reads (50 nt) were generated, of which, 133 million were obtained in AP13 and the rest 135 million in VS16. More than 90% of these reads were mapped to the switchgrass reference genome (V1.1). We identified 6619 and 5369 differentially expressed genes in VS16 and AP13, respectively. Gene ontology and KEGG pathway analysis identified key genes that regulate important pathways including C4 photosynthesis, photorespiration and phenylpropanoid metabolism. A series of genes (33) involved in photosynthetic pathway were up-regulated in AP13 but only two genes showed higher expression in VS16. We identified three dicarboxylate transporter homologs that were highly expressed in AP13. Additionally, genes that mediate drought, heat, and salinity tolerance were also identified. Vesicular transport proteins, syntaxin and signal recognition particles were seen to be up-regulated in VS16. Analyses of selected genes involved in biosynthesis of secondary metabolites, plant-pathogen interaction, membrane transporters, heat, drought and salinity stress responses confirmed significant variation in the relative expression reflected in RNA-Seq data between VS16 and AP13 genotypes. The phenylpropanoid pathway genes identified here are potential targets for biofuel conversion., Competing Interests: The authors declare that they have no conflict of interest.

The genome sequence of Medicago truncatula was published and released in 2011. A Tnt1 insertional mutant population with 21,700 independently regenerated lines was completed in 2012 at The Samuel Roberts Noble Foundation. With an estimated 25 insertions per line, the Tnt1 mutant population harbors more than 500,000 insertions in the M. truncatula genome. Based on the genome size, average gene length, and random insertion of Tnt1into the genome, the mutant population affects about 90% of genes in the M. truncatula genome. Therefore, the mutant population enables functional characterization of most genes in the M. truncatula genome. From 2006 to 2011, we sequenced about 33,000 flanking sequence tags (FSTs) from 2600 Tnt1 lines using TAIL-PCR followed by TA cloning, plasmid isolation, and traditional Sanger sequencing. To accelerate FST sequencing, we developed a two-dimensional DNA pooling strategy coupled with next-generation sequencing and produced about 380,000 FSTs from all 21,700 lines in a relatively short time. All FSTs are BLAST searchable in a web-based database. One can quickly search the database to find M. truncatula mutant lines with Tnt1 insertions in most genes of interest.

Natural plant microbiomes are abundant and have a remarkably robust composition, both as epiphytes on the plant surface and as endophytes within plant tissues. Microbes in the former "habitat" face limited nutrients and harsh environmental conditions, while those in the latter likely lead a more sheltered existence. The most populous and diverse of these microbiomes are associated with the zone around the plant roots, commonly referred to as the rhizosphere. A majority of recent studies characterize these plant-associated microbiomes by community profiling of bacteria and fungi, using amplicon-based marker genes and next-generation sequencing (NGS). Here, we collate a group of protocols that incorporate current best practices and optimized methodologies for sampling, handling of samples, and rRNA library preparation for variable regions of V5-V6 and V9 of the bacterial 16S ribosomal RNA (rRNA) gene, and the ITS2 region joining the 5.8S and 28S regions of the fungal rRNA gene. Samples collected for such culture-independent analyses can also be used for the actual isolation of microbes of interest, perhaps even those identified by the libraries described above. One group of microbes that holds promise for mediating plant stress incurred by drought are bacteria that are capable of reducing or eliminating the plant's perception of the stress through degradation of the gaseous plant hormone ethylene, which is abundantly produced in response to drought stimuli. This is accomplished by some types of soil bacteria that can produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which is the immediate precursor to ethylene. Here we provide a high-throughput protocol for screening of ACC deaminase-producing bacteria for the applied purpose of mitigating the impact of plant drought stress.