Correction to "Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics".

[This corrects the article DOI: 10.1021/acssuschemeng.2c01160.].

Leaf layer-based transcriptome profiling for discovery of epidermal-selective promoters in Medicago truncatula.

MAIN CONCLUSION: Transcriptomics of manually dissected leaf layers from Medicago truncatula identifies genes with preferential expression in upper and/or lower epidermis. The promoters of these genes confer epidermal-specific expression of transgenes. Improving the quality and quantity of proanthocyanidins (PAs) in forage legumes has potential to improve the nitrogen nutrition of ruminant animals and protect them from the risk of pasture bloat, as well as parasites. However, ectopic constitutive accumulation of PAs in plants by genetic engineering can significantly inhibit growth. We selected the leaf epidermis as a candidate tissue for targeted engineering of PAs or other pathways. To identify gene promoters selectively expressed in epidermal tissues, we performed comparative transcriptomic analyses in the model legume Medicago truncatula, using five tissue samples representing upper epidermis, lower epidermis, whole leaf without upper epidermis, whole leaf without lower epidermis, and whole leaf. We identified 52 transcripts preferentially expressed in upper epidermis, most of which encode genes involved in flavonoid biosynthesis, and 53 transcripts from lower epidermis, with the most enriched category being anatomical structure formation. Promoters of the preferentially expressed genes were cloned from the M. truncatula genome and shown to direct tissue-selective promoter activities in transient assays. Expression of the PA pathway transcription factor TaMYB14 under control of several of the promoters in transgenic alfalfa resulted in only modest MYB14 transcript accumulation and low levels of PA production. Activity of a subset of promoters was confirmed by transcript analysis in field-grown alfalfa plants throughout the growing season, and revealed variable but consistent expression, which was generally highest 3-4 weeks after cutting. We conclude that, although the selected promoters show acceptable tissue-specificity, they may not drive high enough transcription factor expression to activate the PA pathway.

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor.

BACKGROUND: The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. RESULTS: We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. CONCLUSIONS: Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls.

Development and commercialization of reduced lignin alfalfa.

Reducing lignin content in forage legumes can improve digestibility and, correspondingly, animal performance, and alfalfa (Medicago sativa) is the first genetically engineered crop commercialized for improved forage digestibility. Lignin reduction was achieved by downregulating the gene encoding caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), and development of the commercial product, branded as HarvXtra, required the coordination of two research institutions and two companies, and more than 15 years of research and field trials. Lignin modification has positive impacts on forage management. Future developments will likely stack lignin modification with additional forage quality traits.

Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula.

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.

Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.).

Genes encoding seven enzymes of the monolignol pathway were independently downregulated in alfalfa (Medicago sativa) using antisense and/or RNA interference. In each case, total flux into lignin was reduced, with the largest effects arising from the downregulation of earlier enzymes in the pathway. The downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase, hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate 5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional changes in lignin and wall-bound hydroxycinnamic acids consistent with the current models of the monolignol pathway. However, downregulating caffeoyl CoA 3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin composition differed in plants downregulated in either cinnamate 4-hydroxylase or phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were observed in the various transgenic lines. The current model for monolignol and ferulate biosynthesis appears to be an over-simplification, at least in alfalfa, and additional enzymes may be needed for the 3-O-methylation reactions of S lignin and ferulate biosynthesis.

Nitrogen fixation by white lupin under phosphorus deficiency.

BACKGROUND AND AIMS: White lupin is highly adapted to growth in a low-P environment. The objective of the present study was to evaluate whether white lupin grown under P-stress has adaptations in nodulation and N2 fixation that facilitate continued functioning. METHODS: Nodulated plants were grown in silica sand supplied with N-free nutrient solution containing 0 to 0.5 mm P. At 21 and 37 d after inoculation (DAI) growth, nodulation, P and N concentration, N2 fixation (15N2 uptake and H2 evolution), root/nodule net CO2 evolution and CO2 fixation (14CO2 uptake) were measured. Furthermore, at 21 DAI in-vitro activities and transcript abundance of key enzymes of the C and N metabolism in nodules were determined. Moreover, nodulation in cluster root zones was evaluated. KEY RESULTS: Treatment without P led to a lower P concentration in shoots, roots, and nodules. In both treatments, with or without P, the P concentration in nodules was greater than that in the other organs. At 21 DAI nitrogen fixation rates did not differ between treatments and the plants displayed no symptoms of P or N deficiency on their shoots. Although nodule number at 21 DAI increased in response to P-deficiency, total nodule mass remained constant. Increased nodule number in P-deficient plants was associated with cluster root formation. A higher root/nodule CO2 fixation in the treatment without P led to a lower net CO2 release per unit fixed N, although the total CO2 released per unit fixed N was higher in the treatment without P. The higher CO2 fixation was correlated with increased transcript abundance and enzyme activities of phosphoenolpyruvate carboxylase and malate dehydrogenase in nodules. Between 21 and 37 DAI, shoots of plants grown without P developed symptoms of N- and P-deficiency. By 37 DAI the P concentration had decreased in all organs of the plants treated with no P. At 37 DAI, nitrogen fixation in the treatment without P had almost ceased. CONCLUSIONS: Enhanced nodulation in cluster root zones and increased potential for organic acid production in root nodules appear to contribute to white lupin's resilience to P-deficiency.

A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa).

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene.

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22-24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.