Unveiling unique alternative splicing responses to low temperature in Zoysia japonica through ZjRTD1.0, a high-quality reference transcript dataset.

Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity. This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low-temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low-temperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica.

KINASE-INDUCIBLE DOMAIN INTERACTING 8 regulates helical pod morphology in Medicago truncatula.

Leguminosae exhibits a wide diversity of legume forms with varying degrees of spiral morphologies, serving as an ideal clade for studying the growth and development of spiral organs. While soybean (Glycine max) develops straight pods, the pod of the model legume Medicago truncatula is a helix structure. Despite the fascinating structures and intensive description of the pods in legumes, little is known regarding the genetic mechanism underlying the highly varied spirality of the legume pods. In this study, we found that KINASE-INDUCIBLE DOMAIN INTERACTING 8 (MtKIX8) plays a key role in regulating the pod structure and spirality in M. truncatula. Unlike the coiled and barrel-shaped helix pods of the wild type, the pods of the mtkix8 mutant are loose and deformed and lose the topologic structure as observed in the wild-type pods. In the pods of the mtkix8 mutant, the cells proliferate more actively and overly expand, particularly in the ventral suture, resulting in uncoordinated growth along the dorsal and ventral sutures of pods. The core cell cycle genes CYCLIN D3s are upregulated in the mtkix8 pods, leading to the prolonged growth of the ventral suture region of the pods. Our study revealed the key role of MtKIX8 in regulating seed pod development in M. truncatula and demonstrates a genetic regulatory model underlying the establishment of the helical pod in legumes.

Natural variation of GmFATA1B regulates seed oil content and composition in soybean.

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl-acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.

MOTHER-OF-FT-AND-TFL1 regulates the seed oil and protein content in soybean.

Soybean is a major crop that produces valuable seed oil and protein for global consumption. Seed oil and protein are regulated by complex quantitative trait loci (QTLs) and have undergone intensive selections during the domestication of soybean. It is essential to identify the major genetic components and understand their mechanism behind seed oil and protein in soybean. We report that MOTHER-OF-FT-AND-TFL1 (GmMFT) is the gene of a classical QTL that has been reported to regulate seed oil and protein content in many studies. Mutation of MFT decreased seeds oil content and weight in both Arabidopsis and soybean, whereas increased expression of GmMFT enhanced seeds oil content and weight. Haplotype analysis showed that GmMFT has undergone selection, which resulted in the extended haplotype homozygosity in the cultivated soybean and the enriching of the oil-favorable allele in modern soybean cultivars. This work unraveled the GmMFT-mediated mechanism regulating seed oil and protein content and seed weight, and revealed a previously unknown function of MFT that provides new insights into targeted soybean improvement and breeding.

Bioengineering Techniques to Improve Nitrogen Transformation and Utilization: Implications for Nitrogen Use Efficiency and Future Sustainable Crop Production.

Nitrogen (N) is crucial for plant growth and development, especially in physiological and biochemical processes such as component of different proteins, enzymes, nucleic acids, and plant growth regulators. Six categories, such as transporters, nitrate absorption, signal molecules, amino acid biosynthesis, transcription factors, and miscellaneous genes, broadly encompass the genes regulating NUE in various cereal crops. Herein, we outline detailed research on bioengineering modifications of N metabolism to improve the different crop yields and biomass. We emphasize effective and precise molecular approaches and technologies, including N transporters, transgenics, omics, etc., which are opening up fascinating opportunities for a complete analysis of the molecular elements that contribute to NUE. Moreover, the detection of various types of N compounds and associated signaling pathways within plant organs have been discussed. Finally, we highlight the broader impacts of increasing NUE in crops, crucial for better agricultural yield and in the greater context of global climate change.

Genome-Wide Identification, Characterization, and Expression Analysis of SPIRAL1 Family Genes in Legume Species.

The SPIRAL1 (SPR1) gene family encodes microtubule-associated proteins that are essential for the anisotropic growth of plant cells and abiotic stress resistance. Currently, little is known about the characteristics and roles of the gene family outside of Arabidopsis thaliana. This study intended to investigate the SPR1 gene family in legumes. In contrast to that of A. thaliana, the gene family has undergone shrinking in the model legume species Medicago truncatula and Glycine max. While the orthologues of SPR1 were lost, very few SPR1-Like (SP1L) genes were identified given the genome size of the two species. Specifically, the M. truncatula and G. max genomes only harbor two MtSP1L and eight GmSP1L genes, respectively. Multiple sequence alignment showed that all these members contain conserved N- and C-terminal regions. Phylogenetic analysis clustered the legume SP1L proteins into three clades. The SP1L genes showed similar exon-intron organizations and similar architectures in their conserved motifs. Many essential cis-elements are present in the promoter regions of the MtSP1L and GmSP1L genes associated with growth and development, plant hormones, light, and stress. The expression analysis revealed that clade 1 and clade 2 SP1L genes have relatively high expression in all tested tissues in Medicago and soybean, suggesting their function in plant growth and development. MtSP1L-2, as well as clade 1 and clade 2 GmSP1L genes, display a light-dependent expression pattern. The SP1L genes in clade 2 (MtSP1L-2, GmSP1L-3, and GmSP1L-4) were significantly induced by sodium chloride treatment, suggesting a potential role in the salt-stress response. Our research provides essential information for the functional studies of SP1L genes in legume species in the future.

Identification of GOLDEN2-like transcription factor genes in soybeans and their role in regulating plant development and metal ion stresses.

The Golden 2-Like (G2-like or GLK) transcription factors are essential for plant growth, development, and many stress responses as well as heavy metal stress. However, G2-like regulatory genes have not been studied in soybean. This study identified the genes for 130 G2-Like candidates' in the genome of Glycine max (soybean). These GLK genes were located on all 20 chromosomes, and several of them were segmentally duplicated. Most GLK family proteins are highly conserved in Arabidopsis and soybean and were classified into five major groups based on phylogenetic analysis. These GmGLK gene promoters share cis-acting elements involved in plant responses to abscisic acid, methyl jasmonate, auxin signaling, low temperature, and biotic and abiotic stresses. RNA-seq expression data revealed that the GLK genes were classified into 12 major groups and differentially expressed in different tissues or organs. The co-expression network complex revealed that the GmGLK genes encode proteins involved in the interaction of genes related to chlorophyll biosynthesis, circadian rhythms, and flowering regulation. Real-time quantitative PCR analysis confirmed the expression profiles of eight GLK genes in response to cadmium (Cd) and copper (Cu) stress, with some GLK genes significantly induced by both Cd and Cu stress treatments, implying a functional role in defense responsiveness. Thus, we present a comprehensive perspective of the GLK genes in soybean and emphasize their important role in crop development and metal ion stresses.

Developing Genetic Engineering Techniques for Control of Seed Size and Yield.

Many signaling pathways regulate seed size through the development of endosperm and maternal tissues, which ultimately results in a range of variations in seed size or weight. Seed size can be determined through the development of zygotic tissues (endosperm and embryo) and maternal ovules. In addition, in some species such as rice, seed size is largely determined by husk growth. Transcription regulator factors are responsible for enhancing cell growth in the maternal ovule, resulting in seed growth. Phytohormones induce significant effects on entire features of growth and development of plants and also regulate seed size. Moreover, the vegetative parts are the major source of nutrients, including the majority of carbon and nitrogen-containing molecules for the reproductive part to control seed size. There is a need to increase the size of seeds without affecting the number of seeds in plants through conventional breeding programs to improve grain yield. In the past decades, many important genetic factors affecting seed size and yield have been identified and studied. These important factors constitute dynamic regulatory networks governing the seed size in response to environmental stimuli. In this review, we summarized recent advances regarding the molecular factors regulating seed size in Arabidopsis and other crops, followed by discussions on strategies to comprehend crops' genetic and molecular aspects in balancing seed size and yield.

A perspective on the molecular mechanism in the control of organ internal (IN) asymmetry during petal development.

Floral zygomorphy (monosymmetry) is a key innovation in flowering plants and is related to the coevolution of plants and their animal pollinators. The molecular basis underlying floral zygomorphy has been analysed, and two regulatory pathways have been identified: one determines the dorsoventral (DV) asymmetry along the floral plan, and the other controls organ internal (IN) asymmetry during petal development. While strides have been made to understand the molecular mechanism controlling DV asymmetry, which mainly involves an interplay between TCP and MYB transcription factors, the molecular pathway regulating IN asymmetry remains largely unknown. In this review, we discuss what is known about regulators and the molecular pathway regulating IN asymmetry. Our analysis revealed that the regulation of IN asymmetry occurs at the cellular, tissue, and organ genesis levels during petal development and that the regulatory mechanism is likely integrated into different developmental paths, such as floral and root nodule development. Although the molecular regulation of IN asymmetry is not be a linear path, a key hub for the regulatory network could be vascular patterning during petal organogenesis.

Tannin tolerance lactic acid bacteria screening and their effects on fermentation quality of stylo and soybean silages.

Some excellent legume forages are difficult to ensile naturally due to their high buffering capacity and low water-soluble carbohydrate content. This may cause serious problems like proteolysis. In the present study, strains of lactic acid bacteria with high acid productivity and high tannin tolerance were screened from different silages and combined with tannic acid (TA) as an addition to ensiling. The screened strains were identified as Lactobacillus plantarum (LP), with four of these strains then selected for their high tannin tolerance. Stylosanthes guianensis and whole-plant soybean (WPS) were ensiled with 1 and 2% (fresh matter basis) TA, four LP strains alone (6 log10 colony forming units per gram of fresh matter), or TA combined with LP strains. Fermentation parameters and in vitro rumen fermentation characteristics were analyzed after 30 days of fermentation. The results showed that TA + LP can be used to reduce pH values (P < 0.01), non-protein nitrogen (P < 0.01), and ammonia-nitrogen (P < 0.01). The in vitro crude protein digestibility of WPS silage was also decreased with the addition of TA + LP (P < 0.01). These results indicate that the addition of TA combined with tannin tolerance LP strains may improve the fermentation quality of legume silage, especially for reducing proteolysis.

Salt-tolerant endophytic bacterium Enterobacter ludwigii B30 enhance bermudagrass growth under salt stress by modulating plant physiology and changing rhizosphere and root bacterial community.

Osmotic and ionic induced salt stress suppresses plant growth. In a previous study, Enterobacter ludwigii B30, isolated from Paspalum vaginatum, improved seed germination, root length, and seedling length of bermudagrass (Cynodon dactylon) under salt stress. In this study, E. ludwigii B30 application improved fresh weight and dry weight, carotenoid and chlorophyll levels, catalase and superoxide dismutase activities, indole acetic acid content and K+ concentration. Without E. ludwigii B30 treatment, bermudagrass under salt stress decreased malondialdehyde and proline content, Y(NO) and Y(NPQ), Na+ concentration, 1-aminocyclopropane-1-carboxylate, and abscisic acid content. After E. ludwigii B30 inoculation, bacterial community richness and diversity in the rhizosphere increased compared with the rhizosphere adjacent to roots under salt stress. Turf quality and carotenoid content were positively correlated with the incidence of the phyla Chloroflexi and Fibrobacteres in rhizosphere soil, and indole acetic acid (IAA) level was positively correlated with the phyla Actinobacteria and Chloroflexi in the roots. Our results suggest that E. ludwigii B30 can improve the ability of bermudagrass to accumulate biomass, adjust osmosis, improve photosynthetic efficiency and selectively absorb ions for reducing salt stress-induced injury, while changing the bacterial community structure of the rhizosphere and bermudagrass roots. They also provide a foundation for understanding how the bermudagrass rhizosphere and root microorganisms respond to endophyte inoculation.

Comprehensive Genomic Survey, Evolution, and Expression Analysis of GIF Gene Family during the Development and Metal Ion Stress Responses in Soybean.

The GIF gene family is one of the plant transcription factors specific to seed plants. The family members are expressed in all lateral organs produced by apical and floral meristems and contribute to the development of leaves, shoots, flowers, and seeds. This study identified eight GIF genes in the soybean genome and clustered them into three groups. Analyses of Ka/Ks ratios and divergence times indicated that they had undergone purifying selection during species evolution. RNA-sequence and relative expression patterns of these GmGIF genes tended to be conserved, while different expression patterns were also observed between the duplicated GIF members in soybean. Numerous cis-regulatory elements related to plant hormones, light, and stresses were found in the promoter regions of these GmGIF genes. Moreover, the expression patterns of GmGIF members were confirmed in soybean roots under cadmium (Cd) and copper (Cu) stress, indicating their potential functions in the heavy metal response in soybean. Our research provides valuable information for the functional characterization of each GmGIF gene in different legumes in the future.

A Survey of Enhanced Cold Tolerance and Low-Temperature-Induced Anthocyanin Accumulation in a Novel Zoysia japonica Biotype.

Zoysia japonica is a warm-season turfgrass that is extensively used in landscaping, sports fields, and golf courses worldwide. Uncovering the low-temperature response mechanism of Z. japonica can help to accelerate the development of new cold-tolerant cultivars, which could be used to prolong the ornamental and usage duration of turf. A novel Z. japonica biotype, YueNong-9 (YN-9), was collected from northeastern China for this study. Phenotypic measurements, cold-tolerance investigation, and whole-transcriptome surveys were performed on YN-9 and LanYin-3 (LY-3), the most popular Z. japonica cultivar in Southern China. The results indicated the following: YN-9 has longer second and third leaves than LY-3; when exposed to the natural low temperature during winter in Guangzhou, YN-9 accumulated 4.74 times more anthocyanin than LY-3; after cold acclimation and freezing treatment, 83.25 ± 9.55% of YN-9 survived while all LY-3 leaves died, and the dark green color index (DGCI) value of YN-9 was 1.78 times that of LY-3; in YN-9, there was a unique up-regulation of Phenylalanine ammonia-lyase (PAL), Homeobox-leucine Zipper IV (HD-ZIP), and ATP-Binding Cassette transporter B8 (ABCB8) expressions, as well as a unique down-regulation of zinc-regulated transporters and iron-regulated transporter-like proteins (ZIPs) expression, which may promote anthocyanin biosynthesis, transport, and accumulation. In conclusion, YN-9 exhibited enhanced cold tolerance and is thus an excellent candidate for breeding cold-tolerant Z. japonica variety, and its unique low-temperature-induced anthocyanin accumulation and gene responses provide ideas and candidate genes for the study of low-temperature tolerance mechanisms and genetic engineering breeding.

Rapid identification of mutations caused by fast neutron bombardment in Medicago truncatula.

BACKGROUND: Fast neutron bombardment (FNB) is a very effective approach for mutagenesis and has been widely used in generating mutant libraries in many plant species. The main type of mutations of FNB mutants are deletions of DNA fragments ranging from few base pairs to several hundred kilobases, thus usually leading to the null mutation of genes. Despite its efficiency in mutagenesis, identification of the mutation sites is still challenging in many species. The traditional strategy of positional cloning is very effective in identifying the mutation but time-consuming. With the availability of genome sequences, the array-based comparative genomic hybridization (CGH) method has been developed to detect the mutation sites by comparing the signal intensities of probes between wild-type and mutant plants. Though CGH method is effective in detecting copy number variations (CNVs), the resolution and coverage of CGH probes are not adequate to identify mutations other than CNVs. RESULTS: We report a new strategy and pipeline to sensitively identify the mutation sites of FNB mutants by combining deep-coverage whole-genome sequencing (WGS), polymorphism calling, and customized filtering in Medicago truncatula. Initially, we performed a bulked sequencing for a FNB white nodule (wn) mutant and its wild-type like plants derived from a backcross population. Following polymorphism calling and filtering, validation by manual check and Sanger sequencing, we identified that SymCRK is the causative gene of white nodule mutant. We also sequenced an individual FNB mutant yellow leaves 1 (yl1) and wild-type plant. We identified that ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN 1 (EGY1) is the candidate gene for M. truncatula yl1 mutant. CONCLUSION: Our results demonstrated that the method reported here is rather robust in identifying the mutation sites for FNB mutants.

Effects of Gallic Acid on Fermentation Parameters, Protein Fraction, and Bacterial Community of Whole Plant Soybean Silage.

Whole plant soybean (WPS) is a kind of legume resource with characteristics of high nutrition, large biomass, and wide distribution. In the present study, we have investigated the feasibility and effects of gallic acid (GA) on WPS silage quality, nitrogen distribution, tannin content, and bacterial community. The 0.5 and 1% (fresh matter basis) GA were added into WPS for dynamic ensiling (days 3, 7, 14, and 30, respectively). The results showed that the WPS silage with GA addition significantly decreased pH value (6.16-5.38 at ensiling day 30), coliform bacteria count and butyric acid (65.3-62.0 g/kg dry matter at ensiling day 30), and amino nitrogen contents (259-88.2 g/kg total nitrogen at ensiling day 30) and promoted lactic acid (9.62-31.5 g/kg dry matter at ensiling day 30), acetic acid (24.1-85.6 g/kg dry matter at ensiling day 30), and tannin (total phenol and hydrolyzable tannin) contents. Additionally, the GA addition also contributed to the change of bacterial community, where Firmicutes and Lactobacillus were most abundant on phylum and genus levels, respectively. The above results suggested that GA additive applied in WPS silage was an effective strategy to protect nutrition and improve fermentation quality, and the 1% GA addition showed a better effect.

Circadian rhythms driving a fast-paced root clock implicate species-specific regulation in Medicago truncatula.

Plants have a hierarchical circadian structure comprising multiple tissue-specific oscillators that operate at different speeds and regulate the expression of distinct sets of genes in different organs. However, the identity of the genes differentially regulated by the circadian clock in different organs, such as roots, and how their oscillations create functional specialization remain unclear. Here, we profiled the diurnal and circadian landscapes of the shoots and roots of Medicago truncatula and identified the conserved regulatory sequences contributing to transcriptome oscillations in each organ. We found that the light-dark cycles strongly affect the global transcriptome oscillation in roots, and many clock genes oscillate only in shoots. Moreover, many key genes involved in nitrogen fixation are regulated by circadian rhythms. Surprisingly, the root clock runs faster than the shoot clock, which is contrary to the hierarchical circadian structure showing a slow-paced root clock in both detached and intact Arabidopsis thaliana (L.) Heynh. roots. Our result provides important clues about the species-specific circadian regulatory mechanism, which is often overlooked, and possibly coordinates the timing between shoots and roots independent of the current prevailing model.

Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

BACKGROUND: C2H2 zinc finger proteins (C2H2 ZFPs) play vital roles in shaping many aspects of plant growth and adaptation to the environment. Plant genomes harbor hundreds of C2H2 ZFPs, which compose one of the most important and largest transcription factor families in higher plants. Although the C2H2 ZFP gene family has been reported in several plant species, it has not been described in the model leguminous species Medicago truncatula. RESULTS: In this study, we identified 218 C2H2 type ZFPs with 337 individual C2H2 motifs in M. truncatula. We showed that the high rate of local gene duplication has significantly contributed to the expansion of the C2H2 gene family in M. truncatula. The identified ZFPs exhibit high variation in motif arrangement and expression pattern, suggesting that the short C2H2 zinc finger motif has been adopted as a scaffold by numerous transcription factors with different functions to recognize cis-elements. By analyzing the public expression datasets and quantitative RT-PCR (qRT-PCR), we identified several C2H2 ZFPs that are specifically expressed in certain tissues, such as the nodule, seed, and flower. CONCLUSION: Our genome-wide work revealed an expanded C2H2 ZFP gene family in an important legume M. truncatula, and provides new insights into the diversification and expansion of C2H2 ZFPs in higher plants.

LAZY Gene Family in Plant Gravitropism.

Adapting to the omnipresent gravitational field was a fundamental basis driving the flourishing of terrestrial plants on the Earth. Plants have evolved a remarkable capability that not only allows them to live and develop within the Earth's gravity field, but it also enables them to use the gravity vector to guide the growth of roots and shoots, in a process known as gravitropism. Triggered by gravistimulation, plant gravitropism is a highly complex, multistep process that requires many organelles and players to function in an intricate coordinated way. Although this process has been studied for several 100 years, much remains unclear, particularly the early events that trigger the relocation of the auxin efflux carrier PIN-FORMED (PIN) proteins, which presumably leads to the asymmetrical redistribution of auxin. In the past decade, the LAZY gene family has been identified as a crucial player that ensures the proper redistribution of auxin and a normal tropic response for both roots and shoots upon gravistimulation. LAZY proteins appear to be participating in the early steps of gravity signaling, as the mutation of LAZY genes consistently leads to altered auxin redistribution in multiple plant species. The identification and characterization of the LAZY gene family have significantly advanced our understanding of plant gravitropism, and opened new frontiers of investigation into the novel molecular details of the early events of gravitropism. Here we review current knowledge of the LAZY gene family and the mechanism modulated by LAZY proteins for controlling both roots and shoots gravitropism. We also discuss the evolutionary significance and conservation of the LAZY gene family in plants.