Sugarcane is a vital crop with significant economic and industrial value. However, the cultivated sugarcane’s ultra-complex genome still needs to be resolved due to its high ploidy and extensive recombination between the two subgenomes. Here, we generate a chromosomal-scale, haplotype-resolved genome assembly for a hybrid sugarcane cultivar ZZ1. This assembly contains 10.4 Gb genomic sequences and 68,509 annotated genes with defined alleles in two sub-genomes distributed in 99 original and 15 recombined chromosomes. RNA-seq data analysis shows that sugar accumulation-associated gene families have been primarily expanded from the ZZSO subgenome. However, genes responding to pokkah boeng disease susceptibility have been derived dominantly from the ZZSS subgenome. The region harboring the possible smut resistance genes has expanded significantly. Among them, the expansion of WAK and FLS2 families is proposed to have occurred during the breeding of ZZ1. Our findings provide insights into the complex genome of hybrid sugarcane cultivars and pave the way for future genomics and molecular breeding studies in sugarcane.

Miscanthus is a leading bioenergy crop with enormous lignocellulose production potential for biofuels and chemicals. However, lignocellulose recalcitrance leads to biomass process difficulty for an efficient bioethanol production. Hence, it becomes essential to identify the integrative impact of lignocellulose recalcitrant factors on cellulose accessibility for biomass enzymatic hydrolysis. In this study, we analyzed four typical pairs of Miscanthus accessions that showed distinct cell wall compositions and sorted out three major factors that affected biomass saccharification for maximum bioethanol production. Among the three optimal (i.e., liquid hot water, H2SO4 and NaOH) pretreatments performed, mild alkali pretreatment (4% NaOH at 50 °C) led to almost complete biomass saccharification when 1% Tween-80 was co-supplied into enzymatic hydrolysis in the desirable Miscanthus accessions. Consequently, the highest bioethanol yields were obtained at 19% (% dry matter) from yeast fermentation, with much higher sugar–ethanol conversion rates by 94–98%, compared to the other Miscanthus species subjected to stronger pretreatments as reported in previous studies. By comparison, three optimized pretreatments distinctively extracted wall polymers and specifically altered polymer features and inter-linkage styles, but the alkali pretreatment caused much increased biomass porosity than that of the other pretreatments. Based on integrative analyses, excellent equations were generated to precisely estimate hexoses and ethanol yields under various pretreatments and a hypothetical model was proposed to outline an integrative impact on biomass saccharification and bioethanol production subjective to a predominate factor (CR stain) of biomass porosity and four additional minor factors (DY stain, cellulose DP, hemicellulose X/A, lignin G-monomer). Using four pairs of Miscanthus samples with distinct cell wall composition and varied biomass saccharification, this study has determined three main factors of lignocellulose recalcitrance that could be significantly reduced for much-increased biomass porosity upon optimal pretreatments. It has also established a novel standard that should be applicable to judge any types of biomass process technology for high biofuel production in distinct lignocellulose substrates. Hence, this study provides a potential strategy for precise genetic modification of lignocellulose in all bioenergy crops.

Genetic modification of plant cell walls has been implemented to reduce lignocellulosic recalcitrance for biofuel production. Plant glycoside hydrolase family 9 (GH9) comprises endo-β-1,4-glucanase in plants. Few studies have examined the roles of GH9 in cell wall modification. In this study, we independently overexpressed two genes from GH9B subclasses (OsGH9B1 and OsGH9B3) and examined cell wall features and biomass saccharification in transgenic rice plants. Compared with the wild type (WT, Nipponbare), the OsGH9B1 and OsGH9B3 transgenic rice plants, respectively, contained much higher OsGH9B1 and OsGH9B3 protein levels and both proteins were observed in situ with nonspecific distribution in the plant cells. The transgenic lines exhibited significantly increased cellulase activity in vitro than the WT. The OsGH9B1 and OsGH9B3 transgenic plants showed a slight alteration in three wall polymer compositions (cellulose, hemicelluloses, and lignin), in their stem mechanical strength and biomass yield, but were significantly decreased in the cellulose degree of polymerization (DP) and lignocellulose crystalline index (CrI) by 21–22%. Notably, the crude cellulose substrates of the transgenic lines were more efficiently digested by cellobiohydrolase (CBHI) than those of the WT, indicating the significantly increased amounts of reducing ends of β-1,4-glucans in cellulose microfibrils. Finally, the engineered lines generated high sugar yields after mild alkali pretreatments and subsequent enzymatic hydrolysis, resulting in the high bioethanol yields obtained at 22.5% of dry matter. Overproduction of OsGH9B1/B3 enzymes should have specific activity in the postmodification of cellulose microfibrils. The increased reducing ends of β-1,4-glucan chains for reduced cellulose DP and CrI positively affected biomass enzymatic saccharification. Our results demonstrate a potential strategy for genetic modification of cellulose microfibrils in bioenergy crops.

Rice is a major food crop containing large amounts of lignocellulose residues usable for biofuels. In this study, we collected transgenic rice plants that over-produced Trichoderma reesei β-1,4-D-glucosidase (BGL I) into the cell walls in the mature straws. Without any pretreatment, the transgenic rice straws showed a consistently higher biomass enzymatic saccharification than the wild-type (WT) cultivar, in particular when 1% Tween-80 or 0.5% PEG-4000 was co-supplied into the enzymatic hydrolysis. Notably, under mild alkali pretreatment (1% NaOH at 50 °C for 2 h), the desirable transgenic line exhibited complete biomass enzymatic hydrolysis, resulting in the highest bioethanol yield of 21% (% dry matter) when compared with the rice and other bioenergy crops subjected to stronger pretreatment conditions reported in previous studies. Meanwhile, despite relatively low hexose yields obtained under 1% H₂SO₄ pretreatment, the transgenic rice straw also showed high bioethanol production at 18% due to an almost complete sugar–ethanol conversion rate. Chemical analyses indicated that the transgenic rice straw had significantly increased biomass porosity and reduced cellulose features (CrI, DP), which contributed to the largely enhanced biomass enzymatic hydrolysis. In addition, the raised arabinose level in hemicellulose and the lignin H-monomer proportion may also positively affect the biomass enzymatic saccharification in the transgenic rice straw. Hence, this study demonstrated a cost-effective and green lignocellulose conversion technology for high bioethanol production in the transgenic rice straw. It also provided a strategy for the potential genetic modification of plant cell walls in bioenergy crops.

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling. Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R 2/R 2 cv/R 2 ev and RPD values for a perfect prediction capacity. Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.