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Growing concerns over food insecurity and ecosystems health related to population growth and climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In this review, we discuss the domestication of the oilseed-producing cover crop pennycress, which along with the development of other new crops and improvements to farming practices can provide sustainable solutions to address malnutrition and environmental impacts of production agriculture. We highlight some of the new technologies such as bioinformatics-enabled next-generation sequencing and CRISPR genome editing in combination with traditional mutation breeding that has accelerated pennycress development as a new crop and a potential model system. Furthermore, we provide a brief overview of the technologies that can be integrated for improving pennycress and other crops and the status of pennycress development using these technologies.

This article comments on: Tsogtbaatar E, Cocuron J-C, Alonso AP. 2020. Non-conventional pathways enable pennycress (Thlaspi arvense L.) embryos to achieve high biosynthetic efficiency. Journal of Experimental Botany 71, 3037–3051.

The hydroxycinnamic acids p‐coumaric acid (pCA) and ferulic acid (FA) add diversity to the portfolio of products produced by using grass‐fed lignocellulosic biorefineries. The level of lignin‐bound pCA in Zea mays was modified by the alteration of p‐coumaroyl‐CoA monolignol transferase expression. The biomass was processed in a lab‐scale alkaline‐pretreatment biorefinery process and the data were used for a baseline technoeconomic analysis to determine where to direct future research efforts to couple plant design to biomass utilization processes. It is concluded that future plant engineering efforts should focus on strategies that ramp up accumulation of one type of hydroxycinnamate (pCA or FA) predominantly and suppress that of the other. Technoeconomic analysis indicates that target extraction titers of one hydroxycinnamic acid need to be >50 g kg−1 biomass, at least five times higher than observed titers for the impure pCA/FA product mixture from wild‐type maize. The technical challenge for process engineers is to develop a viable process that requires more than 80 % reduction of the isolation costs., Stream on: A competitive lignocellulosic biorefinery will need to produce multiple product streams that include liquid fuel, solid fuel, and commodity chemicals. In this study, some key parameters are identified for the extraction of the valuable commodity chemicals p‐coumaric acid and ferulic acid from one of the waste streams in an alkaline‐pretreatment‐based biorefinery.

The oilseed species Thlaspi arvense (pennycress)—a weed that was only recently removed from the wild—has the potential to provide new sources of food and bioproducts when grown as a winter cover crop. Domestication of wild species has historically taken hundreds to thousands of years, but by making use of large-scale high-throughput comparative gene and phenotype analyses, along with recently developed technological tools, it has been possible to greatly accelerate this process. By taking advantage of extensive gene and phenotype knowledge in the related plant Arabidopsis, mutations for early maturity, reduced pod shatter, reduced seed glucosinolates and improved fatty acid composition were identified. Progress has been made to rapidly stack these traits in order to domesticate the plant, allowing it to fit within current crop cycles and to have improved seed harvestability and nutritional content. Pennycress, domesticated as a winter cover crop, may provide new sources of food, animal feed and bioproducts—and solutions to food security. Thlaspi arvense (pennycress) has the potential to provide new sources of food and bioproducts when grown as a winter cover crop. Here, Chopra et al. demonstrate that multiple desirable traits can be stacked to rapidly domesticate pennycress. The resulting crop integrates into current crop rotations and produces seeds with improved nutritional qualities, easier harvesting and suitability for human consumption.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Thlaspi arvense (pennycress) has the potential for domestication as a new oilseed crop. Information from an extensive body of research on the related plant species Arabidopsis can be used to greatly speed this process. Genome-scale comparisons in this paper documented that pennycress and Arabidopsis share similar gene duplication. This finding led to the hypothesis that it should be possible to isolate Arabidopsis-like mutants in pennycress. This proved to be true, as forward genetic screens identified floral and vegetative pennycress mutants that were similar to mutants found in Arabidopsis. Extending this approach, it was shown that most of the pennycress genes responsible for the formation of oxidized tannins could be rapidly identified. The causative mutations in the pennycress mutants could be identified either by PCR amplification of candidate genes or through whole-genome sequencing (WGS) analysis. In all, WGS was used to characterize 95 ethyl methane sulfonate mutants, which revealed a mutation rate of 4.09 mutations per megabase. A sufficient number of non-synonymous mutations were identified to create a mutant gene index that could be used for reverse genetic approaches to identify pennycress mutants of interest. As proof of concept, a Ta-max3-like dwarf mutant and Ta-kcs5/cer60-like wax mutants deficient in the biosynthesis of long chain fatty acids were identified. Overall, these studies demonstrate that translational genomics can be used to promote the domestication of pennycress. Furthermore, the ease with which important findings could be made in pennycress makes this species a new potential model plant.

Summary To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML‐FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML‐FA production are as follows: (1) The presence of an active FERULOYL‐CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16‐fold increase in lignin‐bound ML‐FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p‐COUMAROYL‐CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p‐coumaroyl‐CoA intermediate did not change ML‐FA incorporation, nor did partial loss of CINNAMOYL‐CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt‐1 mutant resulted in a 32‐fold increase in ML‐FA incorporation into lignin over the wild‐type level.

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana. We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyonPAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant. IMPORTANCE Although the role of plant defense mechanisms against viruses are relatively well studied in dicots and in incompatible plant-microbe interactions, studies of their roles in compatible interactions and in grasses are lagging behind. In this study, we leveraged the emerging grass model Brachypodium and genetic resources to dissect Panicum mosaic virus (PMV)- and its satellite virus (SPMV)-compatible grass-virus interactions. We found a significant role for PAL1 in the production of salicylic acid (SA) in response to PMV/SPMV infections and that SA is an essential component of the defense response preventing the plant from succumbing to viral infection. Our results suggest a convergent role for the SA defense pathway in both compatible and incompatible plant-virus interactions and underscore the utility of Brachypodium for grass-virus biology.

Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of seeds rich in triacylglycerols and protein, making it an attractive target for domestication into an offseason oilseed cash cover crop. Pennycress is easily genetically transformed, enabling synthetic biology approaches to tailor oil properties for specific biofuel and industrial applications. To test the feasibility in pennycress of producing TAGs and acetyl-TAGs rich in medium-chain fatty acids (MCFAs; C6–C14) for industrial, biojet fuel and improved biodiesel applications, we generated transgenic lines with seed-specific expression of unique acyltransferase (LPAT and diacylglycerol acyltransferase) genes and thioesterase (FatB) genes isolated from Cuphea viscosissima, Cuphea avigera var. pulcherrima, Cuphea hookeriana, Coco nucifera, and Umbellularia californica. Wild-type pennycress seed TAGs accumulate no fatty acids shorter than 16C and less than 5 mol percent C16 as palmitic acid (16:0). Co-expressing UcFatB and CnLPAT produced up to 17 mol% accumulation of lauric acid (12:0) in seed TAGs, whereas CvFatB1 CvLPAT2 CpDGAT1 combinatorial expression produced up to 27 mol% medium chain FAs Medium Chain Fatty Acids mostly in the form of capric acid (10:0). CpFatB2 ChFatB2 combinatorial expression predominantly produced, in equal parts, up to 28 mol% myristic acid (14:0) and palmitic acid. Genetically crossing the combinatorial constructs into a fatty acid elongation1 (fae1) mutant that produced no 22:1 erucic acid, and with an Euonymus alatus diacylglycerol acetyltransferase (EaDAcT)-expressing line that produced 60 mol% acetyl-TAGs, had no or relatively minor effects on MCFAs accumulation, suggesting fluxes to MCFAs were largely unaltered. Seed germination assays revealed no or minor delays in seed germination for most lines, the exception being CpFatB2 ChFatB2-expressing lines, which had substantially slower seed germination rates. Taken together, these data show that pennycress can be engineered to produce seeds accumulating modest amounts of MCFAs of varying carbon-chain length in TAGs and acetyl-TAGs, with rates of seed germination being delayed in only some cases. We hypothesize that increasing MCFAs further may require functional reductions to endogenous transferases and/or other FA elongases.

Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

During the phototropic response, plant cells reorient their cortical microtubule arrays by generating many new growing microtubules through severing. This study shows how new plus ends created by severing, which lack GTP-caps, are stabilized by CLASP to allow for microtubule amplification and array reorientation., Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP’s function in promoting microtubule amplification by severing and array reorientation.

Summary Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run‐off on otherwise barren farmland. We demonstrate that pennycress can serve as a user‐friendly model system akin to Arabidopsis that is well‐suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring‐type Spring 32‐10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter‐type pennycress, as well as predominantly a one‐to‐one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium‐mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR‐Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low‐viscosity acetyl‐triacylglycerol‐containing seed oil suitable as a diesel‐engine drop‐in fuel. Adoption of pennycress as a model system will accelerate oilseed‐crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands.

The oilseed speciesThlaspi arvense(pennycress) is being domesticated as a new crop that can provide both important ecosystem services and intensify farmland output. Through the use of high throughput sequencing and phenotyping, along with classical mutagenesis key traits needed for pennycress domestication have been identified. Domestication traits identified herein include reduced pod shatter, early maturity, reduced seed glucosinolate levels, and improved oil fatty acid content. By taking advantage of pennycress’ close genetic relationship withArabidopsis thaliana, the causative mutations responsible for each of these traits have been identified. These mutations have been used to develop molecular markers to begin to stack the traits into individual lines.

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. As severing produces new plus ends behind the stabilizing GTP-cap, an important and unanswered question is how these are stabilizedin vivoto promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing and find that CLASP is required for rapid cortical array reorientation. Inclaspmutants both rescue of shrinking plus ends and the regrowth of plus ends immediately after severing are reduced, computational modeling reveals that it is the specific stabilization of severed ends that explains CLASP’s function in promoting microtubule amplification by severing and cortical array reorientation.

Background The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA. Results Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Conclusions Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0793-1) contains supplementary material, which is available to authorized users.

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.

© 2016 Petrik, Cass, Padmakshan, Foster, Vogel, Karlen, Ralph and Sedbrook. Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

Plants have convergently evolved to use monolignol ferulate conjugates to produce lignins containing chemically labile backbone esters., Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl–coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.

Diversification of agroecological systems to enhance agrobiodiversity is likely to be critical to advancing environmental, economic, and social sustainability of agriculture. Temperate-zone agroecological systems that are currently organized for production of summer-annual crops can be diversified by integration of fallow-season and perennial crops. Integration of such crops can improve sustainability of these agroecological systems, with minimal interference with current agricultural production. Importantly, these crops can provide feedstocks for a wide range of new bio-products that are forming a new agricultural bioeconomy, potentially providing greatly increased economic incentives for diversification. However, while there are many fallow-season and perennial crops that might be used in such a “bioeconomic” strategy for diversification, most are not yet well adapted and highly-marketable. Efforts are underway to enhance adaptation and marketability of many such crops. Critically, these efforts require a strategic approach that addresses the inherent complexity of these projects. We outline a suitable approach, which we term “sustainable commercialization”: a coordinated innovation process that integrates a new crop into the agriculture of a region, while intentionally addressing economic, environmental and social sustainability challenges via multi-stakeholder governance. This approach centers on a concerted effort to coordinate and govern innovation in three critical areas: germplasm development, multifunctional agroecosystem design and management, and development of end uses, supply chains, and markets. To exemplify the approach, we describe an ongoing effort to commercialize a new fallow-season crop, field pennycress (Thlaspi arvense L.).

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.

Microtubule associated proteins (MAPs) are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.

Highlight Reducing the function of PAL, the first enzyme in the phenylpropanoid pathway, in Brachypodium distachyon alters cell wall composition, increases fungal susceptibility, but minimally affects caterpillar herbivory and abiotic stress tolerance., The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

Wild-type Arabidopsis (Arabidopsis thaliana L. Heynh.) roots growing on a tilted surface of impenetrable hard-agar media adopt a wave-like pattern and tend to skew to the right of the gravity vector (when viewed from the back of the plate through the medium). Reversible root-tip rotation often accompanies the clockwise and counterclockwise curves that form each wave. These rotations are manifested by epidermal cell file rotation (CFR) along the root. Loss-of-function alleles of ROOT HAIR DEFECTIVE3 (RHD3), a gene previously implicated in the control of vesicle trafficking between the endoplasmic reticulum and the Golgi compartments, resulted in an almost complete suppression of epidermal CFR, root skewing, and waving on hard-agar surfaces. Several other root hair defective mutants (rhd2-1, rhd4-1, and rhd6-1) did not exhibit dramatic alterations in these root growth behaviors, suggesting that a generalized defect in root hair formation is not responsible for the surface-dependent phenotypes of rhd3. However, similar alterations in root growth behavior were observed in a variety of mutants characterized by defects in cell expansion (cob-1, cob-2, eto1-1, eto2-1, erh2-1, and erh3-1). The erh2-1 and rhd3-1 mutants differed from other anisotropic cell expansion mutants, though, by an inability to respond to low doses of the microtubule-binding drug propyzamide, which normally causes enhanced left-handed CFR and right skewing. We hypothesize that RHD3 may control epidermal CFR, root skewing, and waving on hard-agar surfaces by regulating the traffic of wall- or plasma membrane-associated determinants of anisotropic cell expansion.

Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth.

To investigate how roots respond to directional cues, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

Oilseed crops are sources of oils and seed meal having a multitude of uses. While the domestication of soybean and rapeseed took extended periods of time, new genome-based techniques have ushered in an era where crop domestication can occur rapidly. One attractive target for rapid domestication is the winter annual plant Field Pennycress (Thlaspi arvense L.; pennycress; Brassicaceae). Pennycress grows widespread throughout temperate regions of the world and could serve as a winter oilseed-producing cover crop. If grown throughout the USA Midwest Corn Belt, for example, pennycress could produce as much as 840L/ha oils and 1470kg/ha press-cake annually on 16 million hectares of farmland currently left fallow during the fall through spring months. However, wild pennycress strains have inconsistent germination and stand establishment, un-optimized maturity for a given growth zone, suboptimal oils and meal quality for biofuels and food production, and significant harvest loss due to pod shatter. In this review, we describe the virtues and current shortcomings of pennycress and discuss how knowledge from studying Arabidopsis thaliana and other Brassicas, in combination with the advent of affordable next generation sequencing, can bring about the rapid domestication and improvement of pennycress and other crops.

Using the transgenic AEQUORIN system, we showed that the cotyledons and leaves of Arabidopsis thaliana seedlings developed a biphasic luminescence response to anoxia, indicating changes in cytosolic Ca2+ levels. A fast and transient luminescence peak occurred within minutes of anoxia, followed by a second, prolonged luminescence response that lasted 1.5 to 4 h. The Ca2+ channel blockers Gd3+, La3+, and ruthenium red (RR) partially inhibited the first response and promoted a larger and earlier second response, suggesting different origins for these responses. Both Gd3+ and RR also partially inhibited anaerobic induction of alcohol dehydrogenase gene expression. However, although anaerobic alcohol dehydrogenase gene induction occurred in seedlings exposed to water-agar medium and in roots, related luminescence responses were absent. Upon return to normoxia, the luminescence of cotyledons, leaves, and roots dropped quickly, before increasing again in a Gd3+-, La3+-, ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid-, and RR-sensitive fashion.

The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5[prime] promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial [beta]-glucuronidase showed robust [beta]-glucuronidase activity primarily in guard cells.

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

Plant microtubules (MTs) polymerize and depolymerize in a process termed dynamic instability. This allows the assembly, reorganization, and disassembly of at least four MT arrays throughout the cell cycle. The cortical MT array lines the plasma membrane during interphase and plays a central role in directional cell expansion. Microtubule-associated proteins (MAPs) decorate cortical MTs with distinct patterns, regulating MT dynamic instability, MT severing, and other array-ordering processes. The Arabidopsis root has emerged as a highly useful system for identifying and studying cell-expansion-related MAPs. Here, we review how cortical MTs are thought to behave and become ordered in expanding root cells, and we discuss the emerging picture of how MAPs fundamentally govern MT ordering and directional growth processes.

In animals and yeast, CLASP proteins are microtubule plus-end tracking proteins (+TIPS) involved in the regulation of microtubule plus-end dynamics and stabilization. Here we show that mutations in the Arabidopsis CLASP homolog result in various plant growth reductions, cell form defects and reduced mitotic activity. Analysis of Arabidopsis plants that carry a YFP:AtCLASP fusion construct regulated by the AtCLASP native promoter showed similarities to the described localization of the animal CLASP proteins, but also prominent differences including punctate and preferential localization along cortical microtubules. Colocalization studies of YFP:AtCLASP and CFP:EB1b also showed that AtCLASP is enriched at the plus ends of microtubules where it localizes behind the AtEB1b protein. Moreover, AtCLASP overexpression causes abnormal cortical microtubule bundling and array organization. Cortical microtubule arrays have evolved to be prominent in plants, and our findings suggest that plant CLASP proteins may have adopted specific functions in regulating cortical microtubule properties and cell growth.

Triacylglycerol (TAG) is a storage lipid used for food purposes and as a renewable feedstock for biodiesel production. WRINKLED1 (WRI1) is a transcription factor that governs fatty acid (FA) synthesis and, indirectly, TAG accumulation in oil-storing plant tissues, and its ectopic expression has led to TAG accumulation in vegetative tissues of different dicotyledonous plants. The ectopic expression of BdWRI1 in the grass Brachypodium distachyon induced the transcription of predicted genes involved in glycolysis and FA biosynthesis, and TAG content was increased up to 32.5-fold in 8-week-old leaf blades. However, the ectopic expression of BdWRI1 also caused cell death in leaves, which has not been observed previously in dicotyledonous plants such as Arabidopsis ( Arabidopsis thaliana ). Lipid analysis indicated that the free FA content was 2-fold elevated in BdWRI1 -expressing leaf blades of B. distachyon . The transcription of predicted genes involved in β-oxidation was induced. In addition, linoleic FA treatment caused cell death in B. distachyon leaf blades, an effect that was reversed by the addition of the FA biosynthesis inhibitor cerulenin. Taken together, ectopic expression of BdWRI1 in B. distachyon enhances FA biosynthesis and TAG accumulation in leaves, as expected, but also leads to increased free FA content, which has cytotoxic effects leading to cell death. Thus, while WRI appears to ubiquitously affect FA biosynthesis and TAG accumulation in diverse plants, its ectopic expression can lead to undesired side effects depending on the context of the specific lipid metabolism of the respective plant species.

Microtubule-associated proteins (MAPs) serve a wide variety of functions, from constructing and maintaining the microtubule cytoskeleton to using this cytoskeleton to transport cargo and to tether molecules that are involved in numerous cellular processes. Throughout the cell cycle, distinct microtubule arrays carry out specific roles in cytokinesis, karyokinesis, and cell expansion. Recent findings have shed new light on the importance of MAPs in controlling microtubule growth dynamics as well as in cross-linking microtubules to facilitate the formation and function of these cytoskeletal arrays.

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.

ARG1 (ALTERED RESPONSE TO GRAVITY) is required for normal root and hypocotyl gravitropism. Here, we show that targeting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the corresponding organs of arg1-2 null mutants. The cytosolic alkalinization of root cap columella cells that normally occurs very rapidly upon gravistimulation is lacking in arg1-2 mutants. Additionally, vertically grown arg1-2 roots appear to accumulate a greater amount of auxin in an expanded domain of the root cap compared with the wild type, and no detectable lateral auxin gradient develops across mutant root caps in response to gravistimulation. We also demonstrate that ARG1 is a peripheral membrane protein that may share some subcellular compartments in the vesicular trafficking pathway with PIN auxin efflux carriers. These data support our hypothesis that ARG1 is involved early in gravitropic signal transduction within the gravity-perceiving cells, where it influences pH changes and auxin distribution. We propose that ARG1 affects the localization and/or activity of PIN or other proteins involved in lateral auxin transport.

Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, we find that low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. We also describe defects in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, we find that embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis.

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589–1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 ( AGR1 ) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae , AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1 -related genes in Arabidopsis , suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.