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.

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.

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.

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.

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.

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.

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.

Negative gravitropic response of roots directs auxin flow to control root gravitropism.

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.

Increasing seed size and quality by manipulating BIG SEEDS1 in legume species.

Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes.

Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance.

Regulation of compound leaf development by PHANTASTICA in Medicago truncatula.

Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant.

Control of dissected leaf morphology by a Cys(2)His(2) zinc finger transcription factor in the model legume Medicago truncatula.

Plant leaves are diverse in their morphology, reflecting to a large degree the plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leaflets at the tip. We show that development of the trifoliate leaves is determined by the Cys(2)His(2) zinc finger transcription factor PALM1. Loss-of-function mutants of PALM1 develop dissected leaves with five leaflets clustered at the tip. We demonstrate that PALM1 binds a specific promoter sequence and down-regulates the expression of the M. truncatula LEAFY/UNIFOLIATA orthologue SINGLE LEAFLET1 (SGL1), encoding an indeterminacy factor necessary for leaflet initiation. Our data indicate that SGL1 is required for leaflet proliferation in the palm1 mutant. Interestingly, ectopic expression of PALM1 effectively suppresses the lobed leaf phenotype from overexpression of a class 1 KNOTTED1-like homeobox protein in Arabidopsis plants. Taken together, our results show that PALM1 acts as a determinacy factor, regulates the spatial-temporal expression of SGL1 during leaf morphogenesis and together with the LEAFY/UNIFOLIATA orthologue plays an important role in orchestrating the compound leaf morphology in M. truncatula.

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.

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.

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.

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.