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BackgroundSingle guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established.ResultsWe developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 (GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation.ConclusionsThis screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.

Background:Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Results:We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. Conclusion:The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.

BackgroundWe previously developed several strategies to engineer plants to produce cost-efficient biofuels from plant biomass. Engineered Arabidopsis plants with low xylan and lignin content showed normal growth and improved saccharification efficiency under standard growth conditions. However, it remains to be determined whether these engineered plants perform well under drought stress, which is the primary source of abiotic stress in the field.ResultsUpon exposing engineered Arabidopsis plants to severe drought, we observed better survival rates in those with a low degree of xylan acetylation, low lignin, and low xylan content compared to those in wild-type plants. Increased pectic galactan content had no effect on drought tolerance. The drought-tolerant plants exhibited low water loss from leaves, and drought-responsive genes (RD29A, RD29B, DREB2A) were generally up-regulated under drought stress, which did not occur in the well-watered state. When compared with the wild type, plants with low lignin due to expression of QsuB, a 3-dehydroshikimate dehydratase, showed a stronger response to abscisic acid (ABA) in assays for seed germination and stomatal closure. The low-lignin plants also accumulated more ABA in response to drought than the wild-type plants. On the contrary, the drought tolerance in the engineered plants with low xylan content and low xylan acetylation was not associated with differences in ABA content or response compared to the wild type. Surprisingly, we found a significant increase in galactose levels and sugar released from the low xylan-engineered plants under drought stress.ConclusionsThis study shows that plants engineered to accumulate less lignin or xylan are more tolerant to drought and activate drought responses faster than control plants. This is an important finding because it demonstrates that modification of secondary cell walls does not necessarily render the plants less robust in the environment, and it shows that substantial changes in biomass composition can be achieved without compromising plant resilience.

BackgroundGrasses are lignocellulosic materials useful to supply the billion-tons annual requirement for renewable resources that aim to produce transportation fuels and a variety of chemicals. However, the polysaccharides contained in grass cell walls are built in a recalcitrant composite. Deconstruction of these cell walls is still a challenge for the energy-efficient and economically viable transformation of lignocellulosic materials. The varied tissue-specific distribution of cell wall components adds complexity to the origins of cell wall recalcitrance in grasses. This complexity usually led to empirically developed pretreatment processes to overcome recalcitrance. A further complication is that efficient pretreatment procedures generally treat the less recalcitrant tissues more than necessary, which results in the generation of undesirable biomass degradation products.ResultsSix different sugarcane hybrids were used as model grasses to evaluate the tissue-specific distribution of hemicelluloses and the role of these components in cell wall recalcitrance. Acetylated glucuronoarabinoxylan (GAX) occurs in all tissues. Mixed-linkage glucan (MLG) was relevant in the innermost regions of the sugarcane internodes (up to 15.4 % w/w), especially in the low-lignin content hybrids. Immunofluorescence microscopy showed that xylans predominated in vascular bundles, whereas MLG occurred mostly in the parenchyma cell walls from the pith region of the hybrids with low-lignin content. Evaluation of the digestibility of sugarcane polysaccharides by commercial enzymes indicated that the cell wall recalcitrance varied considerably along the internode regions and in the sugarcane hybrids. Pith regions of the hybrids with high MLG and low-lignin contents reached up to 85 % cellulose conversion after 72 h of hydrolysis, without any pretreatment.ConclusionsThe collective characteristics of the internode regions were related to the varied recalcitrance found in the samples. Components such as lignin and GAX were critical for the increased recalcitrance, but low cellulose crystallinity index, high MLG contents, and highly substituted GAX contributed to the generation of a less recalcitrant material.

BackgroundSlow-degrading, fossil fuel-derived plastics can have deleterious effects on the environment, especially marine ecosystems. The production of bio-based, biodegradable plastics from or in plants can assist in supplanting those manufactured using fossil fuels. Polyhydroxybutyrate (PHB) is one such biodegradable polyester that has been evaluated as a possible candidate for relinquishing the use of environmentally harmful plastics.ResultsPHB, possessing similar properties to polyesters produced from non-renewable sources, has been previously engineered in sugarcane, thereby creating a high-value co-product in addition to the high biomass yield. This manuscript illustrates the coupling of a Fourier-transform infrared microspectrometer, equipped with a focal plane array (FPA) detector, with multivariate imaging to successfully identify and localize PHB aggregates. Principal component analysis imaging facilitated the mining of the abundant quantity of spectral data acquired using the FPA for distinct PHB vibrational modes. PHB was measured in the chloroplasts of mesophyll and bundle sheath cells, acquiescent with previously evaluated plant samples.ConclusionThis study demonstrates the power of IR microspectroscopy to rapidly image plant sections to provide a snapshot of the chemical composition of the cell. While PHB was localized in sugarcane, this method is readily transferable to other value-added co-products in different plants.