Large and stable genome edits at the sorghum alpha kafirin locus result in changes in chromatin accessibility and globally increased expression of genes encoding lysine enrichment.

Introduction: Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it's grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus. Methods: A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression. Results: Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor's involvement in the kernel response to reduced prolamins. Discussion: Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum's response to reduced seed storage proteins and the process of proteome rebalancing.

Transformation and gene editing in the bioenergy grass Miscanthus.

BACKGROUND: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. RESULTS: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. CONCLUSIONS: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

Chemical and genetic variation in feral Cannabis sativa populations across the Nebraska climate gradient.

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine transcript abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in female relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis.

A phytobacterial TIR domain effector manipulates NAD+ to promote virulence.

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+ ) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1's suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1's intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1's E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1's TIR domain possesses different catalytic specificity than other TIR domain-containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence.

Development of mPing-based activation tags for crop insertional mutagenesis.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

The LATERAL ROOT DENSITY gene regulates root growth during water stress in wheat.

Drought stress is the major limiting factor in agriculture. Wheat, which is the most widely grown crop in the world, is predominantly cultivated in drought-prone rainfed environments. Since roots play a critical role in water uptake, root response to water limitations is an important component for enhancing wheat adaptation. In an effort to discover novel genetic sources for improving wheat adaptation, we characterized a wheat translocation line with a chromosomal segment from Agropyron elongatum, a wild relative of wheat, which unlike common wheat maintains root growth under limited-water conditions. By exploring the root transcriptome data, we found that reduced transcript level of LATERAL ROOT DENSITY (LRD) gene under limited water in the Agropyron translocation line confers it the ability to maintain root growth. The Agropyron allele of LRD is down-regulated in response to water limitation in contrast with the wheat LRD allele, which is up-regulated by water deficit stress. Suppression of LRD expression in wheat RNAi plants confers the ability to maintain root growth under water limitation. We show that exogenous gibberellic acid (GA) promotes lateral root growth and present evidence for the role of GA in mediating the differential regulation of LRD between the common wheat and the Agropyron alleles under water stress. Suppression of LRD also had a positive pleiotropic effect on grain size and number under optimal growth conditions. Collectively, our findings suggest that LRD can be potentially useful for improving wheat response to water stress and altering yield components.

Response of Sorghum Enhanced in Monolignol Biosynthesis to Stalk Rot Pathogens.

To increase phenylpropanoid constituents and energy content in the versatile C4 grass sorghum (Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3-O-methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum-inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina-inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum. Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60, Bmr2, or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Engineering linear, branched-chain triterpene metabolism in monocots.

Triterpenes are thirty-carbon compounds derived from the universal five-carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large-scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T0 mature plants) were obtained using the cytosolic-targeting strategy. Plastid-targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid-targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.

Overexpression of the Sorghum bicolor SbCCoAOMT alters cell wall associated hydroxycinnamoyl groups.

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin. SbCCoAOMT was overexpressed to assess the impact of increasing the amount of this enzyme on biomass composition. SbCCoAOMT overexpression increased both soluble and cell wall-bound (esterified) ferulic and sinapic acids, however lignin concentration and its composition (S/G ratio) remained unaffected. This increased deposition of hydroxycinnamic acids in these lines led to an increase in total energy content of the stover. In stalk and leaf midribs, the increased histochemical staining and autofluorescence in the cell walls of the SbCCoAOMT overexpression lines also indicate increased phenolic deposition within cell walls, which is consistent with the chemical analyses of soluble and wall-bound hydroxycinnamic acids. The growth and development of overexpression lines were similar to wild-type plants. Likewise, RNA-seq and metabolite profiling showed that global gene expression and metabolite levels in overexpression lines were also relatively similar to wild-type plants. Our results demonstrate that SbCCoAOMT overexpression significantly altered cell wall composition through increases in cell wall associated hydroxycinnamic acids without altering lignin concentration or affecting plant growth and development.

Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum.

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.

Overexpression of SbMyb60 in Sorghum bicolor impacts both primary and secondary metabolism.

Few transcription factors have been identified in C4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis.

Validation of QTL mapping and transcriptome profiling for identification of candidate genes associated with nitrogen stress tolerance in sorghum.

BACKGROUND: Quantitative trait loci (QTLs) detected in one mapping population may not be detected in other mapping populations at all the time. Therefore, before being used for marker assisted breeding, QTLs need to be validated in different environments and/or genetic backgrounds to rule out statistical anomalies. In this regard, we mapped the QTLs controlling various agronomic traits in a recombinant inbred line (RIL) population in response to Nitrogen (N) stress and validated these with the reported QTLs in our earlier study to find the stable and consistent QTLs across populations. Also, with Illumina RNA-sequencing we checked the differential expression of gene (DEG) transcripts between parents and pools of RILs with high and low nitrogen use efficiency (NUE) and overlaid these DEGs on to the common validated QTLs to find candidate genes associated with N-stress tolerance in sorghum. RESULTS: An F7 RIL population derived from a cross between CK60 (N-stress sensitive) and San Chi San (N-stress tolerant) inbred sorghum lines was used to map QTLs for 11 agronomic traits tested under different N-levels. Composite interval mapping analysis detected a total of 32 QTLs for 11 agronomic traits. Validation of these QTLs revealed that of the detected, nine QTLs from this population were consistent with the reported QTLs in earlier study using CK60/China17 RIL population. The validated QTLs were located on chromosomes 1, 6, 7, 8, and 9. In addition, root transcriptomic profiling detected 55 and 20 differentially expressed gene (DEG) transcripts between parents and pools of RILs with high and low NUE respectively. Also, overlay of these DEG transcripts on to the validated QTLs found candidate genes transcripts for NUE and also showed the expected differential expression. For example, DEG transcripts encoding Lysine histidine transporter 1 (LHT1) had abundant expression in San Chi San and the tolerant RIL pool, whereas DEG transcripts encoding seed storage albumin, transcription factor IIIC (TFIIIC) and dwarfing gene (DW2) encoding multidrug resistance-associated protein-9 homolog showed abundant expression in CK60 parent, similar to earlier study. CONCLUSIONS: The validated QTLs among different mapping populations would be the most reliable and stable QTLs across germplasm. The DEG transcripts found in the validated QTL regions will serve as future candidate genes for enhancing NUE in sorghum using molecular approaches.

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

Mapping QTLs and association of differentially expressed gene transcripts for multiple agronomic traits under different nitrogen levels in sorghum.

BACKGROUND: Sorghum is an important C4 crop which relies on applied Nitrogen fertilizers (N) for optimal yields, of which substantial amounts are lost into the atmosphere. Understanding the genetic variation of sorghum in response to limited nitrogen supply is important for elucidating the underlying genetic mechanisms of nitrogen utilization. RESULTS: A bi-parental mapping population consisting of 131 recombinant inbred lines (RILs) was used to map quantitative trait loci (QTLs) influencing different agronomic traits evaluated under normal N (100 kg.ha(-1) fertilizer) and low N (0 kg.ha(-1) fertilizer) conditions. A linkage map spanning 1614 cM was developed using 642 polymorphic single nucleotide polymorphisms (SNPs) detected in the population using Genotyping-By-Sequencing (GBS) technology. Composite interval mapping detected a total of 38 QTLs for 11 agronomic traits tested under different nitrogen levels. The phenotypic variation explained by individual QTL ranged from 6.2 to 50.8%. Illumina RNA sequencing data generated on seedling root tissues revealed 726 differentially expressed gene (DEG) transcripts between parents, of which 108 were mapped close to the QTL regions. CONCLUSIONS: Co-localized regions affecting multiple traits were detected on chromosomes 1, 5, 6, 7 and 9. These potentially pleiotropic regions were coincident with the genomic regions of cloned QTLs, including genes associated with flowering time, Ma3 on chromosome 1 and Ma1 on chromosome 6, gene associated with plant height, Dw2 on chromosome 6. In these regions, RNA sequencing data showed differential expression of transcripts related to nitrogen metabolism (Ferredoxin-nitrate reductase), glycolysis (Phosphofructo-2-kinase), seed storage proteins, plant hormone metabolism and membrane transport. The differentially expressed transcripts underlying the pleiotropic QTL regions could be potential targets for improving sorghum performance under limited N fertilizer through marker assisted selection.

Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in Sorghum bicolor.

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses.

Redirection of metabolic flux for high levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds.

Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1∆9) and cis-vaccenic acid (18:1∆11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ∆9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ∆9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ∆9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ∆9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.

Dt2 is a gain-of-function MADS-domain factor gene that specifies semideterminacy in soybean.

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean.

Evaluation of constitutive iron reductase (AtFRO2) expression on mineral accumulation and distribution in soybean (Glycine max. L).

Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg, and Mo), pod walls (Fe, K, P, Cu, and Ni), leaves (Fe, P, Cu, Ca, Ni, and Mg) and seeds (Fe, Zn, Cu, and Ni). Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves, and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and distribution.

Seed shattering in a wild sorghum is conferred by a locus unrelated to domestication.

Suppression of seed shattering was a key step during crop domestication that we have previously suggested to be convergent among independent cereal lineages. Positional, association, expression, and mutant complementation data all implicate a WRKY transcription factor, SpWRKY, in conferring shattering to a wild sorghum relative, Sorghum propinquum. We hypothesize that SpWRKY functions in a manner analogous to Medicago and Arabidopsis homologs that regulate cell wall biosynthesis genes, with low expression toward the end of floral development derepressing downstream cell wall biosynthesis genes to allow deposition of lignin that initiates the abscission zone in the seed-pedicel junction. The recent discovery of a YABBY locus that confers shattering within Sorghum bicolor and other cereals validated our prior hypothesis that some parallel domestication may have been convergent. Ironically, however, the shattering allele of SpWRKY appears to be recently evolved in S. propinquum and illustrates a case in which the genetic control of a trait in a wild relative fails to extrapolate even to closely related crops. Remarkably, the SpWRKY and YABBY loci lie only 300 kb apart and may have appeared to be a single genetic locus in some sorghum populations.