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. Graphical Abstract

Sweet clover (Melilotus albus) is a high-quality leguminous forage grass with salinity tolerance, drought tolerance, and cold hardiness. We selected four varieties of sweet clover with different sensitivities (061898, 061930, No. 55 white flower, and Ningxia white flower) and analyzed the effects of different concentrations of three sodium salts (Na₂CO₃, NaHCO₃, and NaCl) on their physiology and biochemistry responses. Growth and development indexes (such as germination rate, root length, shoot length), chlorophyll content, osmotic regulators (proline, soluble sugar), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined under saline–alkali stress. Seed germination and seedling growth of all four clover species were significantly inhibited under saline–alkali stress. During germination, seed germination rate, root length, and shoot length decreased with increasing saline and alkaline concentration. Under saline–alkali stress, chlorophyll content tended to increase and then decrease, cell damage and death increased, and malondialdehyde, soluble sugar, and proline content tended to increase and then decrease. Moreover, the activities of SOD, POD, and CAT all increased and then decreased. Under Na₂CO₃ stress, the decrease in chlorophyll content of the resistant variety 061898 was less than in the sensitive Ningxia white flower variety. As the concentrations of Na₂CO₃, NaHCO₃, and NaCl increased, the maximum photochemical efficiency of PSII was significantly affected. The resistant 061898 is capable of maintaining higher photosynthetic efficiency. Furthermore, under treatments with the three kinds of saline–alkali solutions, cell damage and death for Ningxia white flower were greater than in 061898. For 061898, the increases in soluble sugar and proline content were greater and the increase in malondialdehyde content was less, while the antioxidant enzyme activities were higher than those in Ningxia white flower. All four sweet clover varieties had higher stress resistance with neutral than with alkaline salts. When stressed by medium to high saline–alkali concentrations, sweet clover seedlings had increased osmotic substance content, enhanced antioxidant enzyme activity, and regulated physiological metabolism. Additionally, sweet clovers regulated the expression of WRKY33, GH3, CYCD3, OXI1, MKK2, MYC2, JAZ, COI1, PYL, PP2C, TGA, and MPK3 to adapt to the saline–alkali environment and improve saline–alkali tolerance. Our analysis of the sweet clover salinity tolerance mechanism contributes to its further use and is of significant importance for addressing land salinization and promoting sustainable agricultural and pastoral practices in China.

Alfalfa species Medicago sativa L. (MS) and Medicago falcata L. (MF), globally prominent perennial leguminous forages, hold substantial economic value. However, our comprehension of the molecular mechanisms governing their resistance to cold stress remains limited. To address this knowledge gap, we scrutinized and compared MS and MF cold-stress responses at the molecular level following 24 h and 120 h low-temperature exposure (4 °C). Our study revealed that MF had superior physiological resilience to cold stress compared with MS, and its morphology was healthier under cold stress, and its malondialdehyde content and superoxide dismutase activity increased, first, and then decreased, while the soluble sugar content continued to accumulate. Transcriptome analysis showed that after 120 h of exposure, there were different gene-expression patterns between MS and MF, including 1274 and 2983 genes that were continuously up-regulated, respectively, and a total of 923 genes were included, including star cold-resistant genes such as ICE1 and SIP1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed numerous inter-species differences in sustained cold-stress responses. Notably, MS-exclusive genes included a single transcription factor (TF) gene and several genes associated with a single DNA repair-related pathway, whereas MF-exclusive genes comprised nine TF genes and genes associated with 14 pathways. Both species exhibited high-level expression of genes encoding TFs belonging to AP2-EREBP, ARR-B, and bHLH TF families, indicating their potential roles in sustaining cold resistance in alfalfa-related species. These findings provide insights into the molecular mechanisms governing cold-stress responses in MS and MF, which could inform breeding programs aimed at enhancing cold-stress resistance in alfalfa cultivars.

Background: Salt-alkali stress is an abiotic stress that inhibits crop growth and reduces yield. It significantly affects various physiological processes in plants, including photosynthesis, osmotic regulation, and antioxidant defense. However, studies on the transcriptional response mechanisms of Medicago falcata L. under salt-alkali stress are limited. In this study, RNA-seq technology was used to analyze differentially expressed genes (DEGs) in salt-alkali tolerant M.falcata breeding lines (LM18) and the salt-alkali sensitive Hulunbeier (HL) under salt-alkali stress. Furthermore, physiological indicators such as chlorophyll content, proline accumulation, and superoxide dismutase (SOD) activity were assessed to compare the responses of LM18 and HL to salt-alkali stress. By integrating transcriptomic and physiological analyses, this study provides new insights into the physiological and molecular regulatory mechanisms of M. falcata in response to salt-alkali stress. Results: The results showed that compared to the untreated controls, 10,289 and 2,478 DEGs were detected in LM18 and HL M.falcata seedlings, with 788 shared DEGs detected in both. GO functional analysis classified these DEGs into three categories: Biological Process, Cellular Components, and Molecular Functions, with significant enrichment in GO terms such as “response to osmotic stress”, “intramolecular oxidoreductase activity” and “antioxidant activity”. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the involvement of these DEGs in key metabolic pathways, including “Phenylpropanoid biosynthesis”, “Plant hormone signal transduction”, “Plant-pathogen interaction”, “Isoflavonoid biosynthesis”, “Circadian rhythm-plant” and “Photosynthesis—antenna proteins”. Physiological indicators and membership function analysis confirmed that LM18 has greater salt-alkali tolerance than HL. Transcription factor analysis identified 42 transcription factor families, with the ERF family being the most abundant, followed by MYB-related, WRKY, bHLH, and MYB families. Weighted Gene Co-expression Network Analysis (WGCNA) showed that the MEturquoise module exhibited a significant positive correlation with salt-alkali stress and several physiological indicators. Module gene network analysis and GO enrichment revealed that MS.gene64536(MYBP), MS.gene76249(SRM1) and MS.gene049843 (MPK3) have functions related to “response to salt stress” and “positive regulation of response to salt stress”, suggesting their key roles in salt-alkali tolerance in M.falcata. All three genes were upregulated in the salt-alkali tolerant LM18. Conclusions: The GO terms and KEGG pathways significantly enriched in LM18 involved a significantly higher number of DEGs compared to HL, suggesting a more robust and effective mechanism in LM18. These findings highlight the robust molecular and physiological adaptations of LM18 in response to salt-alkali stress.

Alfalfa is a perennial herbaceous forage legume that is significantly and adversely affected by monocropping. Crop rotation is the most effective measure to overcome continuous cropping obstacles. However, the mechanisms of how bacterial communities are affected and the potential links between these effects and cropping systems remain poorly understood. Based on a long-term field experiments with continuous alfalfa and forage crops with alfalfa rotation in the black soil region of the western Songnen Plain in Northeast China, the alterations in soil bacterial community structure using high-throughput sequencing of the 16S rRNA gene and soil chemical properties and enzyme activities were analyzed. The alfalfa–forage oats–silage maize–alfalfa and alfalfa–silage maize–forage oats–alfalfa system significantly increase the levels of total phosphorus and available phosphorus, and promote the activities of acid phosphatase, β-glucosidase, leucine aminopeptidase, and N-acetyl-β-glucosaminidase in comparison to continuous alfalfa. While alfalfa crop rotation did not affect the α-diversity of soil bacteria, it significantly altered the bacterial community composition and structure. Some key taxa are significantly enriched in the crop rotation system soils, including Bacillus, Sphingobium, Paenibacillus, Hydrogenispora, Rubrobacter, Haliangium, and Rubellimicrobium. Additionally, crop rotation with alfalfa increased the stability and complexity of the soil bacterial co-occurrence network. Based on our findings, we recommend promoting the alfalfa–forage oats–silage maize–alfalfa and alfalfa–silage maize–forage oats–alfalfa rotation systems as ideal practices for overcoming the challenges associated with continuous cropping of alfalfa. These systems not only enhance soil nutrient content and enzyme activities but also foster a beneficial microbial community, ultimately improving soil functionality and crop performance.

Assessing the nutritional quality of silage maize (Zea mays L.) hinges largely on its oil content, a complex quantitative trait influenced by multiple genes. Mining candidate genes within oil content-related quantitative trait loci (QTLs) can provide genetic resources and a theoretical foundation for cultivating high-oil silage maize varieties. This study employed 274 doubled haploid (DH) lines derived from the parental lines BY4944 and DNF34-2 to perform main gene plus polygene mixed genetic analysis and complex interval mapping (CIM), with the goal of pinpointing oil content-related QTLs and genes distributed across the Z. mays L. genome. Leveraging 5400 single nucleotide polymorphism (SNPs), a high-resolution silage maize genetic linkage map covering 3864.51 cM was constructed with an average interval between markers of 0.74 cM. Analysis of the map revealed 13 oil content-related QTLs. The most significant large-effect QTL (qOIL-1-1), located on chromosome 1 within the region spanning 240.93 Mb to 256.57 Mb, exhibited a logarithm of odds (LOD) score of 3.34 and explained 5.06% of oil content-related phenotypic variation. Within these QTLs, 617 genes were annotated. Through transcriptome analysis combined with quantitative real-time polymerase chain reaction (RT-qPCR), five candidate genes potentially associated with oil content were predicted and subsequently validated within these genetic loci. This research underscores the potential of identifying candidate genes to enhance breeding efforts aimed at augmenting oil content, thereby advancing animal husbandry practices.