Microalgae biofuels: illuminating the path to a sustainable future amidst challenges and opportunities.

The development of microalgal biofuels is of significant importance in advancing the energy transition, alleviating food pressure, preserving the natural environment, and addressing climate change. Numerous countries and regions across the globe have conducted extensive research and strategic planning on microalgal bioenergy, investing significant funds and manpower into this field. However, the microalgae biofuel industry has faced a downturn due to the constraints of high costs. In the past decade, with the development of new strains, technologies, and equipment, the feasibility of large-scale production of microalgae biofuel should be re-evaluated. Here, we have gathered research results from the past decade regarding microalgae biofuel production, providing insights into the opportunities and challenges faced by this industry from the perspectives of microalgae selection, modification, and cultivation. In this review, we suggest that highly adaptable microalgae are the preferred choice for large-scale biofuel production, especially strains that can utilize high concentrations of inorganic carbon sources and possess stress resistance. The use of omics technologies and genetic editing has greatly enhanced lipid accumulation in microalgae. However, the associated risks have constrained the feasibility of large-scale outdoor cultivation. Therefore, the relatively controllable cultivation method of photobioreactors (PBRs) has made it the mainstream approach for microalgae biofuel production. Moreover, adjusting the performance and parameters of PBRs can also enhance lipid accumulation in microalgae. In the future, given the relentless escalation in demand for sustainable energy sources, microalgae biofuels should be deemed a pivotal constituent of national energy planning, particularly in the case of China. The advancement of synthetic biology helps reduce the risks associated with genetically modified (GM) microalgae and enhances the economic viability of their biofuel production.

Telomere-to-telomere genome of the allotetraploid legume Sesbania cannabina reveals transposon-driven subgenome divergence and mechanisms of alkaline stress tolerance.

Alkaline soils pose an increasing problem for agriculture worldwide, but using stress-tolerant plants as green manure can improve marginal land. Here, we show that the legume Sesbania cannabina is very tolerant to alkaline conditions and, when used as a green manure, substantially improves alkaline soil. To understand genome evolution and the mechanisms of stress tolerance in this allotetraploid legume, we generated the first telomere-to-telomere genome assembly of S. cannabina spanning ∼2,087 Mb. The assembly included all centromeric regions, which contain centromeric satellite repeats, and complete chromosome ends with telomeric characteristics. Further genome analysis distinguished A and B subgenomes, which diverged approximately 7.9 million years ago. Comparative genomic analysis revealed that the chromosome homoeologs underwent large-scale inversion events (>10 Mb) and a significant, transposon-driven size expansion of the chromosome 5A homoeolog. We further identified four specific alkali-induced phosphate transporter genes in S. cannabina; these may function in alkali tolerance by relieving the deficiency in available phosphorus in alkaline soil. Our work highlights the significance of S. cannabina as a green tool to improve marginal lands and sheds light on subgenome evolution and adaptation to alkaline soils.

Genome evolution and initial breeding of the Triticeae grass Leymus chinensis dominating the Eurasian Steppe.

Leymus chinensis, a dominant perennial grass in the Eurasian Steppe, is well known for its remarkable adaptability and forage quality. Hardly any breeding has been done on the grass, limiting its potential in ecological restoration and forage productivity. To enable genetic improvement of the untapped, important species, we obtained a 7.85-Gb high-quality genome of L. chinensis with a particularly long contig N50 (318.49 Mb). Its allotetraploid genome is estimated to originate 5.29 million years ago (MYA) from a cross between the Ns-subgenome relating to Psathyrostachys and the unknown Xm-subgenome. Multiple bursts of transposons during 0.433-1.842 MYA after genome allopolyploidization, which involved predominantly the Tekay and Angela of LTR retrotransposons, contributed to its genome expansion and complexity. With the genome resource available, we successfully developed a genetic transformation system as well as the gene-editing pipeline in L. chinensis. We knocked out the monocot-specific miR528 using CRISPR/Cas9, resulting in the improvement of yield-related traits with increases in the tiller number and growth rate. Our research provides valuable genomic resources for Triticeae evolutionary studies and presents a conceptual framework illustrating the utilization of genomic information and genome editing to accelerate the improvement of wild L. chinensis with features such as polyploidization and self-incompatibility.

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

Identification of cytochrome P450 gene family and functional analysis of HgCYP33E1 from Heterodera glycines.

The cytochrome P450 (CYP) genes of nematode play a crucial role in the metabolic detoxification of xenobiotics including pesticides. Heterodera glycines, also known as the soybean cyst nematode, is a sedentary endoparasite that infests plant roots, causing high annual economic losses in soybean production regions globally. In this study, we identified 36 CYP genes at a genome-wide level of the H. glycines isolate TN10 using all CYPs from Caenorhabditis elegans as queries. Subsequently, a full-length cDNA of HgCYP33E1 which was significantly up-regulated by the conventional nematicide abamectin was initially cloned from H. glycines. It presented significantly higher expressions in the second-stage juvenile (J2) compared to other parasitic stages of H. glycines. qRT-PCR analysis suggested that the expression of HgCYP33E1 was also xenobiotically induced by soybean root exudate and the metabolites of biocontrol agents. Using RNA interference (RNAi), we investigated the function of HgCYP33E1 in H. glycines parasitism and nematicide selectivity. Compared to the control and dsGFP-treated group, silencing of HgCYP33E1 did not affect the J2 behaviors and the early invasion ability, while it decreased the number of J4s in soybean roots after 18-d inoculation with the dsHgCYP33E1-treated nematodes. In addition, knockdown of HgCYP33E1 in H. glycines resulted in an increase in J2 mortality after 24-h incubation with abamectin compared to the GFP dsRNA-soaked and the control group. These findings revealed the potential role of HgCYP33E1 in the xenobiotic detoxification pathway of H. glycines. Moreover, our data also provided valuable gene information for studying the functions of the CYP family in H. glycines host adaption.

Screening of universal DNA barcodes for identifying grass species of Gramineae.

There is currently international interest in applying DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of four candidate plant DNA barcoding regions [rbcL, matK, trnL-F, and internal transcribed spacer (ITS)] in seven genera of Gramineae including Agropyron, Bromus, Elymus, Elytrigia, Festuca, Leymus, and Lolium. Fourteen accessions were analyzed, and matK and ITS showed the highest species, subspecies, and variety discriminatory power, each resolving 11 accessions. Species discrimination using rbcL and trnL-F was lower, resolving 7 and 8 accessions, respectively. Subspecies and variety discrimination using rbcL and trnL-F could not identify 4 accessions of Agropyron. A technical system can be established using the proposed DNA barcode to rapidly and reliably identify the seven genera of Gramineae. This study serves as a "useful reference" for identifying the genetic diversity of grass germplasm resources. DNA barcoding can be utilized to uncover the relatives of different species within the same family or between different families. It can also be used to determine the related groups of important herbage, turfgrass, and crops and provide crucial background information for discovering excellent genes and improving existing crop varieties.

Comparison of soil microbial community between reseeding grassland and natural grassland in Songnen Meadow.

Microorganisms have important ecological functions in ecosystems. Reseeding is considered as one of the main strategies for preventing grassland degradation in China. However, the response of soil microbial community and diversity to reseeding grassland (RG) and natural grassland (NG) remains unclear, especially in the Songnen Meadow. In this study, the soil microbial community compositions of two vegetation restoration types (RG vs NG) were analyzed using a high-throughput sequencing technique. A total of 23,142 microbial OTUs were detected, phylogenetically derived from 11 known bacterial phyla. Soil advantage categories included Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, which together accounted for > 78% of the all phyla in vegetation restoration. The soil microbial diversity was higher in RG than in NG. Two types of vegetation restoration had significantly different characteristics of soil microbial community (P < 0.001). Based on a molecular ecological network analysis, we found that the network in RG had a longer average path distance and modularity than in NG network, making it more resilient to environment changes. Meanwhile, the results of the canonical correspondence analysis and molecular ecological network analysis showed that soil pH (6.34 ± 0.35 in RG and 7.26 ± 0.28 in NG) was the main factor affecting soil microbial community structure, followed by soil moisture (SM) in the Songnen meadow, China. Besides, soil microbial community characteristics can vary significantly in different vegetation restoration. Thus, we suggested that it was necessary and reasonable for this area to popularize reseeding grassland in the future.

Efficient Generation of CRISPR/Cas9-Mediated Homozygous/Biallelic Medicago truncatula Mutants Using a Hairy Root System.

In the process of acquiring mutants mediated by CRISPR/Cas9, plantlets are often regenerated from both mutated and non-mutated cells in a random manner, which increase the odds of chimeric mutated plant. In general, it's necessary to infect more explants or grow to next generation for the need of generating more biallelic or homozygous mutants. In present study, an efficient way of obtaining biallelic or homozygous mutated lines via fast-growing hairy root system without increasing numbers of infected explants or prolonging sexual propagation generation is reported. The fast growing lateral branches of hair roots are originated deep within the parental root from a small number of founder cells at the periphery, and therefore were employed as a library that classify different editing types in different lateral branches in which the homozygous or biallelic lines were screened. Here, MtPDS was employed in a proof-of-concept experiment to evaluate the efficiency of genome editing with our hairy root system. Homozygous/biallelic mutations were found only 1 of the 20 lines in the 1st generation hairy roots, and 8 lines randomly selected were cultured to obtain their branch roots, homozygous/biallelic mutations were found in 6 of the 8 lines in their branch roots. We also tested the method with MtCOMT gene and got the same result. All of the seedlings regenerated from the homozygous/biallelic hairy root mutation lines of MtPDS displayed albino phenotypes. The entire process from vector design to the recovery of plantlets with homozygous/biallelic mutations took approximately 4.5-6.5 months. The whole process could bring inspiration for efficiently generating homozygous/biallelic mutants through CRISPR/Cas9 system from the hairy root or root system of a chimeric mutated transformants, especially for the rare and endangered plants whose explants sources are very limited or the plants that lack of tissue culture and rapid propagation system.

Genome-Wide Identification, Phylogeny, and Expression Analysis of ARF Genes Involved in Vegetative Organs Development in Switchgrass.

Auxin response factors (ARFs) have been reported to play vital roles during plant growth and development. In order to reveal specific functions related to vegetative organs in grasses, an in-depth study of the ARF gene family was carried out in switchgrass (Panicum virgatum L.), a warm-season C4 perennial grass that is mostly used as bioenergy and animal feedstock. A total of 47 putative ARF genes (PvARFs) were identified in the switchgrass genome (2n = 4x = 36), 42 of which were anchored to the seven pairs of chromosomes and found to be unevenly distributed. Sixteen PvARFs were predicted to be potential targets of small RNAs (microRNA160 and 167). Phylogenetically speaking, PvARFs were divided into seven distinct subgroups based on the phylogeny, exon/intron arrangement, and conserved motif distribution. Moreover, 15 pairs of PvARFs have different temporal-spatial expression profiles in vegetative organs (2nd, 3rd, and 4th internode and leaves), which implies that different PvARFs have specific functions in switchgrass growth and development. In addition, at least 14 pairs of PvARFs respond to naphthylacetic acid (NAA) treatment, which might be helpful for us to study on auxin response in switchgrass. The comprehensive analysis, described here, will facilitate the future functional analysis of ARF genes in grasses.

Lignification of Sheepgrass Internodes at Different Developmental Stages and Associated Alteration of Cell Wall Saccharification Efficiency.

Sheepgrass (Leymus chinensis) is a high-quality cool-season forage crop used as pasture and hay for livestock feeds. The presence of lignin in cell walls, however, impairs forage digestibility of such lignocellulosic feedstock. Here, the structural characterization and cell wall composition of sheepgrass internodes were studied, and a progressive increase in cell wall lignification was observed with internode maturation. Lignin composition analysis further revealed a gradual accumulation of guaiacyl and syringyl lignin units during internode development. Consistently, the transcript abundance of lignin-related genes was upregulated in mature internodes, suggesting their potential roles in lignin biosynthesis. Furthermore, the effects of cell wall composition and lignification extent on biomass saccharification efficiency were examined in sheepgrass. The results showed that lignin content, guaiacyl and syringyl lignin unit levels inversely correlated with cell wall digestibility, indicating that lignin is a crucial obstacle for utilizing sheepgrass feedstock. The baseline information obtained in this work will facilitate establishment, grazing management, harvesting and feedstock utilization of sheepgrass in future.