Genome-Wide Identification, Evolution, and Expression Analyses of AP2/ERF Family Transcription Factors in Erianthus fulvus.

The AP2/ERF transcription factor family is one of the most important gene families in plants and plays a vital role in plant abiotic stress responses. Although Erianthus fulvus is very important in the genetic improvement of sugarcane, there are few studies concerning AP2/ERF genes in E. fulvus. Here, we identified 145 AP2/ERF genes in the E. fulvus genome. Phylogenetic analysis classified them into five subfamilies. Evolutionary analysis showed that tandem and segmental duplication contributed to the expansion of the EfAP2/ERF family. Protein interaction analysis showed that twenty-eight EfAP2/ERF proteins and five other proteins had potential interaction relationships. Multiple cis-acting elements present in the EfAP2/ERF promoter were related to abiotic stress response, suggesting that EfAP2/ERF may contribute to adaptation to environmental changes. Transcriptomic and RT-qPCR analyses revealed that EfDREB10, EfDREB11, EfDREB39, EfDREB42, EfDREB44, EfERF43, and EfAP2-13 responded to cold stress, EfDREB5 and EfDREB42 responded to drought stress, and EfDREB5, EfDREB11, EfDREB39, EfERF43, and EfAP2-13 responded to ABA treatment. These results will be helpful for better understanding the molecular features and biological role of the E. fulvus AP2/ERF genes and lay a foundation for further research on the function of EfAP2/ERF genes and the regulatory mechanism of the abiotic stress response.

A chromosome-level genome assembly for Erianthus fulvus provides insights into its biofuel potential and facilitates breeding for improvement of sugarcane.

Erianthus produces substantial biomass, exhibits a good Brix value, and shows wide environmental adaptability, making it a potential biofuel plant. In contrast to closely related sorghum and sugarcane, Erianthus can grow in degraded soils, thus releasing pressure on agricultural lands used for biofuel production. However, the lack of genomic resources for Erianthus hinders its genetic improvement, thus limiting its potential for biofuel production. In the present study, we generated a chromosome-scale reference genome for Erianthus fulvus Nees. The genome size estimated by flow cytometry was 937 Mb, and the assembled genome size was 902 Mb, covering 96.26% of the estimated genome size. A total of 35 065 protein-coding genes were predicted, and 67.89% of the genome was found to be repetitive. A recent whole-genome duplication occurred approximately 74.10 million years ago in the E. fulvus genome. Phylogenetic analysis showed that E. fulvus is evolutionarily closer to S. spontaneum and diverged after S. bicolor. Three of the 10 chromosomes of E. fulvus formed through rearrangements of ancestral chromosomes. Phylogenetic reconstruction of the Saccharum complex revealed a polyphyletic origin of the complex and a sister relationship of E. fulvus with Saccharum sp., excluding S. arundinaceum. On the basis of the four amino acid residues that provide substrate specificity, the E. fulvus SWEET proteins were classified as mono- and disaccharide sugar transporters. Ortho-QTL genes identified for 10 biofuel-related traits may aid in the rapid screening of E. fulvus populations to enhance breeding programs for improved biofuel production. The results of this study provide valuable insights for breeding programs aimed at improving biofuel production in E. fulvus and enhancing sugarcane introgression programs.

Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei.

Lactobacilli such as Lacticaseibacillus (Lcb) paracasei are generally regarded as safe and health-promoting microbes, and have been widely applied in food and pharmaceutical industries. However, the genetic bases of their beneficial properties were mostly uncertain because of the lack of effective genetic manipulation tools. The type II CRISPR-Cas9 system is the largest family present in lactobacilli, but none of them yet have been developed for genetic modifications. Here, we establish the first endogenous CRISPR-Cas9 genome-editing system in lactobacilli. With a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette, the native CRISPR-Cas9 system was reprogrammed to achieve gene deletion and chromosomal insertion at over 90% efficiency, as well as nucleotide substitution at ≥50% efficiency. We also effectively accomplished deletions of large genomic fragments (5-10 kb) and simultaneous deletion of multiple genes at distal loci, both of which are the first cases in lactobacilli when either endogenous or exogenous CRISPR-Cas systems were employed. In addition, we designed a controllable plasmid-targeting sgRNA expression module and integrated it into the editing plasmid. The all-in-one vector realized gene deletion and plasmid curing at high efficiency (>90%). Collectively, the present study develops a convenient and precise genetic tool in Lcb. paracasei and contributes to the genetics and engineering of lactobacilli.

EfGD: the Erianthus fulvus genome database.

Erianthus fulvus (TaxID: 154759) is a valuable germplasm resource in sugarcane breeding and research and has excellent agronomic traits, such as drought resistance, cold resistance, barren tolerance and high brix. With a stable chromosome number (2n = 20) and a small genome (0.9 Gb), it is an ideal candidate for research on sugarcane. Next-generation sequencing technology has enabled a growing number of studies to focus on genomics. Due to the large amount of omics data available, a centralized platform is necessary for ensuring the consistency, independence and maintainability of these large-scale datasets through storage, analysis and integration. Here, we present a comprehensive database for the E. fulvus genome, EfGD. By using the new high-quality reference genome and its annotations, the EfGD provides the largest whole-genome sequencing reference dataset for E. fulvus, which archives 27 165 protein-coding genes and 55 564 488 SNPs from 202 newly resequenced genomes. Furthermore, we created a user-friendly graphical interface for visualizing genomic diversity, population structure and evolution and provided other tools on an open platform. Database URL: https://efgenome.ynau.edu.cn.

Genome-wide identification and expression analysis of the NAC transcription factor family in Saccharum spontaneum under different stresses.

The NAC (NAM, ATAF1/2, and CUC2) transcription factor family is one of the largest families unique to plants and is involved in plant growth and development, organs, morphogenesis, and stress responses. The NAC family has been identified in many plants. As the main source of resistance genes for sugarcane breeding, the NAC gene family in the wild species Saccharum spontaneum has not been systematically studied. In this study, 115 SsNAC genes were identified in the S. spontaneum genome, and these genes were heterogeneously distributed on 25 chromosomes. Phylogenetic analysis divided the SsNAC family members into 18 subgroups, and the gene structure and conserved motif analysis further supported the phylogenetic classification. Four groups of tandemly duplicated genes and nine pairs of segmentally duplicated genes were detected. The SsNAC gene has different expression patterns at different developmental stages of stems and leaves. Further qRT-PCR analysis showed that drought, low-temperature, salinity, pathogenic fungi, and other stresses as well as abscisic acid (ABA) and methyl jasmonate (MeJA) treatments significantly induced the expression of 12 SsNAC genes, indicating that these genes may play a key role in the resistance of S. spontaneum to biotic and abiotic stresses. In summary, the results from this study provide comprehensive information on the NAC transcription factor family, providing a reference for further functional studies of the SsNAC gene.

Mild water and salt stress improve water use efficiency by decreasing stomatal conductance via osmotic adjustment in field maize.

Water and salt stress often occur simultaneously in heavily irrigated arid agricultural areas, yet they are usually studied in isolation. To understand the physiological bases of water use efficiency (WUE) of field-grown maize (Zea mays) at multi-scales under combined water and salt stress, we investigated the joint effects of water and salt stress on physiology, growth, yield, and WUE of two genotypes (XY335 and ZD958). We measured leaf stomatal conductance (gs), net photosynthesis rate (A) and hydraulic traits, whole-plant growth and water use (ET), and final biomass and grain yield. Leaf osmotic adjustment was a key trait of the physiological differences between XY335 and ZD958 under water and salt stress. Although the responses of the two genotypes were different, mild water and salt stress improved intrinsic water use efficiency (iWUE = A/gs) by (i) decreasing gsvia increasing osmotic adjustment and hydraulic resistance, and (ii) declining A via increasing stomatal limitations rather than reducing photosynthetic capacity. Joint water and salt stress had a synergistic effect on reproductive growth and grain formation of maize. Mild water and salt stress reduced ET, stabilized grain yield, and improved grain WUE via declining gs, maintaining photosynthetic capacity, and improving harvest index. Collectively, our study provides a novel insight into the physiological mechanisms of WUE and demonstrates an approach for the efficient management of water and salt by using a growth stage-based deficit irrigation strategy or/and selecting genotypes with strong osmotic adjustment capacity and high harvest index.

Characterization of a Novel Fructosyltransferase from Lactobacillus crispatus, InuCA, That Attaches to the Cell Surface by Electrostatic Interaction.

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here, we identified a novel FTase, InuCA, from Lactobacillus crispatus, a dominant species in the vaginal microbiota of human. InuCA was characterized by the shortest C terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a series of FOS using sucrose as the substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with the fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will contribute to FTase engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. IMPORTANCE L. crispatus is one of the most important species in human vaginal microbiotas, and its persistence is strongly negatively correlated with vaginal diseases. Our research reveals that a novel inulosucrase, InuCA, is present in L. crispatus. InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chains, which is meaningful for the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface, depending on the environmental pH, and also may be involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.

Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina.

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.