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Acetolactate synthase genes (ALS) have successfully been modified for providing resistance to ALS-inhibiting herbicides in many plant species. Based on sequence and expression analyses, we confirmed VvALS1 as the best functional ALS candidate in grapevine. To develop an ALS-based herbicide selection system for facilitating grape transformation, we firstly evaluated the responses of Vitis vinifera cv Chardonnay callus and young in vitro shoots of Vitis vinifera cv Thompson Seedless to several representative ALS-inhibiting herbicides and found a typical linear response curves to some of the herbicides, including chlorsulfuron and imazapyr belonging to the sulfonylurea or imidazolinone families, respectively. Secondly, we created constructs containing amino acid substitutions in the domains which are known to be critical to herbicide resistance and generated transgenic plants for 3 amino acid substitutions using Agrobacterium-mediated transformation of meristematic bulk tissues of Thompson Seedless. Finally, we showed that ectopic expression of two amino acid substitutions (P191S and P191T) at the N-terminal region and another (W568L) at C-terminal region in VvALS resulted in high resistance to chlorsulfuron or imazapyr herbicides in transgenic in vitro shoots. Our work highlighted the potential use of VvALS mutations imparting herbicide resistance as a selectable marker in grapevine transformation research and as a means in fostering grapevine improvement via cisgenesis, paving the way for developing a selectable co-editing system to facilitate transgene-free gene-editing.

Many white grape cultivars have a nonfunctional

Background The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. Results A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. Conclusions A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits. Electronic supplementary material The online version of this article (10.1186/s13068-017-0991-x) contains supplementary material, which is available to authorized users.

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2β-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

High biomass production and wide adaptation has made switchgrass (Panicum virgatum L.) an important candidate lignocellulosic bioenergy crop. One major limitation of this and other lignocellulosic feedstocks is the recalcitrance of complex carbohydrates to hydrolysis for conversion to biofuels. Lignin is the major contributor to recalcitrance as it limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars. Therefore, genetic manipulation of the lignin biosynthesis pathway is one strategy to reduce recalcitrance. Here, we identified a switchgrass Knotted1 transcription factor, PvKN1, with the aim of genetically engineering switchgrass for reduced biomass recalcitrance for biofuel production. Gene expression of the endogenous PvKN1 gene was observed to be highest in young inflorescences and stems. Ectopic overexpression of PvKN1 in switchgrass altered growth, especially in early developmental stages. Transgenic lines had reduced expression of most lignin biosynthetic genes accompanied by a reduction in lignin content suggesting the involvement of PvKN1 in the broad regulation of the lignin biosynthesis pathway. Moreover, the reduced expression of the Gibberellin 20-oxidase (GA20ox) gene in tandem with the increased expression of Gibberellin 2-oxidase (GA2ox) genes in transgenic PvKN1 lines suggest that PvKN1 may exert regulatory effects via modulation of GA signalling. Furthermore, overexpression of PvKN1 altered the expression of cellulose and hemicellulose biosynthetic genes and increased sugar release efficiency in transgenic lines. Our results demonstrated that switchgrass PvKN1 is a putative ortholog of maize KN1 that is linked to plant lignification and cell wall and development traits as a major regulatory gene. Therefore, targeted overexpression of PvKN1 in bioenergy feedstocks may provide one feasible strategy for reducing biomass recalcitrance and simultaneously improving plant growth characteristics.

Salicornia is a new vegetable crop that can be irrigated with highly saline water, even at salt concentrations equivalent to full-strength seawater. During leafy vegetable cultivation, the onset of the reproductive phase is an undesired phenomenon that reduces yield and quality and prevents year-round cultivation. Knowledge about the regulation of floral induction in the members of the tribe Salicornieae, however, is lacking. To establish year-round cultivation, we studied the flower induction of five Salicornia and two Sarcocornia varieties. Plants were grown under two day lengths, 13.5 h and 18 h, and harvested by a repetitive harvest regime. A 13.5-h day length prevented flower induction in the Israeli Salicornia varieties, but a longer day length was required to prevent flower induction in two species originating from more northern latitudes. The onset of the reproductive phase under suboptimal short day length conditions severely reduced vegetative growth and yields in Salicornia .I nSarcocornia, the repetitive harvest regime prevented flowering, making it a promising candidate for year-round cultivation. Irrigating the plants with full-strength seawater (electrical conductivity 48 dS m−1) vs. water with moderate salinity (electrical conductivity 10 dS m −1 ) did not change the general flowering pattern of the studied Salicornieae members.

The halophyte Salicornia was recently introduced as a fresh vegetable crop that thrives in extreme salt conditions. Two annual Salicornia and two perennial Sarcocornia ecotypes were investigated for yield production and nutritional value in response to different seawater concentrations in the irrigation solution. A harvest schedule based on a three-week cycle gave better productivity than a two-week or a four-week cycle. Total yield declined with increasing percentage of seawater above 50% in the irrigation water, however annual plants had always ca 2–3-fold higher fresh biomass in comparison to their perennial counterparts. Increased percentages of seawater in the irrigation solution had the following effects on ion concentrations in the shoots: no change in Ca2+ and Mg2+, a slight increase in K+, and marked elevations in Na+ and Cl−. Importantly, total polyphenol, β-carotene and ureides, all known for their antioxidant capacities, rose with increasing seawater percentage, findings that indicated improved nutritional values for Salicornia and Sarcocornia irrigated with high concentrations of seawater. Impressively, both the annual Salicornia and the perennial Sarcocornia ecotypes exhibited high total shoot lipid contents of up to 2.41 and 2.06 mg g−1 fresh weight, respectively, which included an omega-3 fraction of 47.6 and 41.2% of the total fatty acid content. Moreover, the high fatty acid content of the annual Salicornia ecotype was not significantly affected by increasing seawater concentrations. In this study, we thus demonstrated the feasibility of cultivating Salicornia and Sarcocornia by applying a multiple harvest system and 100% percentages of seawater in the irrigation water generating economic yields with high nutritional value. The findings also showed that Salicornia and Sarcocornia leafy vegetables may attract additional interest as an alternative source of omega-3 polyunsaturated fatty acids for human consumption, even when the crop irrigated solely with seawater.

The growth of crop plants on full-strength seawater constitutes a major challenge because of the high salt content and the limited availability of essential microelements such as molybdenum. For cultivation of the halophyte Salicornia as seawater-grown crop, the effect of molybdate application on total yield production and the activities of the two molybdenum containing enzymes, nitrate reductase (NR) and xanthine dehydrogenase (XDH) was investigated. Increasing molybdate levels in the growth medium supplemented with nitrate or ammonium enhanced yield during multiple shoot removal. Similarly, NR and XDH activities were enhanced with increasing molybdate, indicating that the activity of both enzymes may play an important role in facilitating yield accumulation. Notably, XDH activity in the roots was high and the levels of ureides were low, whereas in the shoot tips ureides were higher and XDH activity was lower. Considering that XDH is a key enzyme in the biosynthesis of the low C/N ratio ureides, these suggest a source–sink relationship between the roots and shoot tips for efficient transport of root-generated ureides to the young growing shoot tips. Our results imply that the supply of molybdenum to Salicornia grown in seawater enhances plant biomass accumulation by increasing the activities of NR and XDH, thereby stimulating a more efficient remobilization of ureides to the newly grown shoot tips after periodic shoot removal.

The APETALA2/ethylene response factor (AP2/ERF) superfamily of transcription factors (TFs) plays essential roles in the regulation of various growth and developmental programs including stress responses. Members of these TFs in other plant species have been implicated to play a role in the regulation of cell wall biosynthesis. Here, we identified a total of 207 AP2/ERF TF genes in the switchgrass genome and grouped into four gene families comprised of 25 AP2-, 121 ERF-, 55 DREB (dehydration responsive element binding)-, and 5 RAV (related to API3/VP) genes, as well as a singleton gene not fitting any of the above families. The ERF and DREB subfamilies comprised seven and four distinct groups, respectively. Analysis of exon/intron structures of switchgrass AP2/ERF genes showed high diversity in the distribution of introns in AP2 genes versus a single or no intron in most genes in the ERF and RAV families. The majority of the subfamilies or groups within it were characterized by the presence of one or more specific conserved protein motifs. In silico functional analysis revealed that many genes in these families might be associated with the regulation of responses to environmental stimuli via transcriptional regulation of the response genes. Moreover, these genes had diverse endogenous expression patterns in switchgrass during seed germination, vegetative growth, flower development, and seed formation. Interestingly, several members of the ERF and DREB families were found to be highly expressed in plant tissues where active lignification occurs. These results provide vital resources to select candidate genes to potentially impart tolerance to environmental stress as well as reduced recalcitrance. Overexpression of one of the ERF genes (PvERF001) in switchgrass was associated with increased biomass yield and sugar release efficiency in transgenic lines, exemplifying the potential of these TFs in the development of lignocellulosic feedstocks with improved biomass characteristics for biofuels.

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2β-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.