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Foxtail millet is a drought-tolerant cereal and forage crop. The basic leucine zipper (bZIP) gene family plays important roles in regulating plant development and responding to stresses. However, the roles of bZIP genes in foxtail millet remain largely uninvestigated. In this study, 92 members of the bZIP transcription factors were identified in foxtail millet and clustered into ten clades. The expression levels of four SibZIP genes (SibZIP11, SibZIP12, SibZIP41, and SibZIP67) were significantly induced after PEG treatment, and SibZIP67 was chosen for further analysis. The studies showed that ectopic overexpression of SibZIP67 in Arabidopsis enhanced the plant drought tolerance. Detached leaves of SibZIP67 overexpressing plants had lower leaf water loss rates than those of wild-type plants. SibZIP67 overexpressing plants improved survival rates under drought conditions compared to wild-type plants. Additionally, overexpressing SibZIP67 in plants displayed reduced malondialdehyde (MDA) levels and enhanced activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) under drought stress. Furthermore, the drought-related genes, such as AtRD29A, AtRD22, AtNCED3, AtABF3, AtABI1, and AtABI5, were found to be regulated in SibZIP67 transgenic plants than in wild-type Arabidopsis under drought conditions. These data suggested that SibZIP67 conferred drought tolerance in transgenic Arabidopsis by regulating antioxidant enzyme activities and the expression of stress-related genes. The study reveals that SibZIP67 plays a beneficial role in drought response in plants, offering a valuable genetic resource for agricultural improvement in arid environments.

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MAIN CONCLUSION: The SiMBR genes in foxtail millet were identified and studied. Heterologous expression of SiMBR2 in Arabidopsis can improve plant tolerance to drought stress by decreasing the level of reactive oxygen species. Foxtail millet (Setaria italica L.), a C4 crop recognized for its exceptional resistance to drought stress, presents an opportunity to improve the genetic resilience of other crops by examining its unique stress response genes and understanding the underlying molecular mechanisms of drought tolerance. In our previous study, we identified several genes linked to drought stress by transcriptome analysis, including SiMBR2 (Seita.7G226600), a member of the MED25 BINDING RING-H2 PROTEIN (MBR) gene family, which is related to protein ubiquitination. Here, we have identified ten SiMBR genes in foxtail millet and conducted analyses of their structural characteristics, chromosomal locations, cis-acting regulatory elements within their promoters, and predicted transcription patterns specific to various tissues or developmental stages using bioinformatic approaches. Further investigation of the stress response of SiMBR2 revealed that its transcription is induced by treatments with salicylic acid and gibberellic acid, as well as by salt and osmotic stresses, while exposure to high or low temperatures led to a decrease in its transcription levels. Heterologous expression of SiMBR2 in Arabidopsis thaliana enhanced the plant's tolerance to water deficit by reducing the accumulation of reactive oxygen species under drought stress. In summary, this study provides support for exploring the molecular mechanisms associated with drought resistance of SiMBR genes in foxtail millet and contributing to genetic improvement and molecular breeding in other crops.

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MAIN CONCLUSION: In this study, we assembled the first complete mitochondrial genome of Setaria italica and confirmed the multi-branched architecture. The foxtail millet (Setaria italica) holds significant agricultural importance, particularly in arid and semi-arid regions. It plays a pivotal role in diversifying dietary patterns and shaping planting strategies. Although the chloroplast genome of S. italica has been elucidated in recent studies, the complete mitochondrial genome remains largely unexplored. In this study, we employed PacBio HiFi sequencing platforms to sequence and assemble the complete mitochondrial genome. The mitochondrial genome spans a total length of 446,614 base pairs and harbors a comprehensive set of genetic elements, including 33 unique protein-coding genes (PCGs), encompassing 24 unique mitochondrial core genes and 9 variable genes, along with 20 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. Our analysis of mitochondrial PCGs revealed a pronounced codon usage preference. For instance, the termination codon exhibits a marked preference for UAA, while alanine (Ala) exhibits a preference for GCU, and glutamine (Gln) favors CAA. Notably, the maximum Relative Synonymous Codon Usage (RSCU) values for cysteine (Cys) and phenylalanine (Phe) are both below 1.2, indicating a lack of strong codon usage preference for these amino acids. Phylogenetic analyses consistently place S. italica in close evolutionary proximity to Chrysopogon zizanioides, relative to other Panicoideae plants. Collinearity analysis showed that a total of 39 fragments were identified to display homology with both the mitochondrial and chloroplast genomes. A total of 417 potential RNA-editing sites were discovered across the 33 mitochondrial PCGs. Notably, all these editing events involved the conversion of cytosine (C) to uracil (U). Through the employment of PCR validation coupled with Sanger sequencing for the anticipated editing sites of these codons, RNA-editing events were conclusively identified at two specific loci: nad4L-2 and atp6-1030. The results of this study provide a pivotal foundation for advanced genomic breeding research in foxtail millet. Furthermore, they impart essential insights that will be instrumental for forthcoming investigations into the evolutionary and molecular dynamics of Panicoideae species.

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Microbiomes provide multiple life-support functions for plants, including nutrient acquisition and tolerance to abiotic and biotic stresses. Considering the importance of C4 cereal and biofuel crops for food security under climate change conditions, more attention has been given recently to C4 plant microbiome assembly and functions. Here, we review the current status of C4 cereal and biofuel crop microbiome research with a focus on beneficial microbial traits for crop growth and health. We highlight the importance of environmental factors and plant genetics in C4 crop microbiome assembly and pinpoint current knowledge gaps. Finally, we discuss the potential of foxtail millet as a C4 model species and outline future perspectives of C4 plant microbiome research.

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Nitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilizing flag leaf N compared with NNRp-SI114. Post-anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which is potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

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DNA methylation is a highly conserved epigenetic modification involved in many biological processes, including growth and development, stress response, and secondary metabolism. DNA demethylase (DNA-deMTase) genes have been identified in some plant species; however, there are no reports on the identification and analysis of DNA-deMTase genes in Foxtail millet (Setaria italica L.). In this study, seven DNA-deMTases were identified in S. italica. These DNA-deMTase genes were divided into four subfamilies (DML5, DML4, DML3, and ROS1) by phylogenetic and gene structure analysis. Further analysis shows that the physical and chemical properties of these DNA-deMTases proteins are similar, contain the typical conserved domains of ENCO3c and are located in the nucleus. Furthermore, multiple cis-acting elements were observed in DNA-deMTases, including light responsiveness, phytohormone responsiveness, stress responsiveness, and elements related to plant growth and development. The DNA-deMTase genes are expressed in all tissues detected with certain tissue specificity. Then, we investigated the abundance of DNA-deMTase transcripts under abiotic stresses (cold, drought, salt, ABA, and MeJA). The results showed that different genes of DNA-deMTases were involved in the regulation of different abiotic stresses. In total, our findings will provide a basis for the roles of DNA-deMTase in response to abiotic stress.

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Setaria italica is an important crop in China that plays a vital role in the Chinese dietary structure. In the last several decades, high temperature has become the most severe climate issue in the world, which causes great harm to the yield and quality formation of millet. In this study, two main cultivated varieties (ZG2 and AI88) were used to explore the photosynthesis and yield index of the whole plant under heat stress. Results implied that photosynthesis was not inhibited during the heat stress, and that the imbalance in sugar transport between different tissues may be the main factor that affects yield formation. In addition, the expression levels of seven SiSUT and twenty-four SiSWEET members were explored. Sugar transporters were heavily affected during the heat stress. The expression of SiSWEET13a was inhibited by heat stress in the stems, which may play a vital role in sugar transport between different tissues. These results provide new insights into the yield formation of crops under heat stress, which will provide guidance to crop breeding and cultivation.

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OBJECTIVE To investigate the effects of Setaria italica extract on improving insomnia model mice and to explore its potential mechanisms. METHODS The mice were randomly assigned into blank group， model group， positive control group （diazepam， 2.6 mg/kg）， and S. italica extract low-dose， medium-dose and high-dose groups （1.2， 2.4， 4.8 g/kg）， with 10 mice in each group. Except for the blank group， all other groups received intraperitoneal injection of para-chlorophenylalanine （PCPA） to establish the insomnia model. After modeling， the blank group and model group were given a constant volume of normal saline intragastrically， and administration groups were given relevant medicine intragastrically， with a volume of 0.01 mL/g， once a day， for 7 consecutive days. After the administration， the open-field test was conducted to observe the praxiological changes of mice， and to determine the levels of 5-hydroxytryptamine （5-HT） and 5-hydroxyindoleacetic acid （5-HTAA） in the hippocampal tissue， as well as the contents of 5-HT， brain-derived neurotrophic factor （BDNF）， interleukin-2 （IL-2）， IL-6， B-cell lymphoma-2 （Bcl- 2）， and Bcl-2-associated X protein （Bax） in the serum. The expression of phosphoinositide 3-kinase/protein kinase B/nuclear factor- κB （PI3K/Akt/NF-κB） signaling pathway related protein was determined in the hippocampus of mice. RESULTS Compared with the model group， the total exercise time of mice in S. italica extract high-dose group was significantly prolonged， but the total rest time was significantly shortened （P＜0.01）； the number of standing times and modification times were significantly reduced （P＜ 0.01）. The contents of 5-HT， BDNF， and Bcl-2 in serum， and Bcl-2/Bax were significantly increased， while the contents of IL-2， IL-6， and Bax were significantly reduced （P＜0.05 or P＜ 0.01）. The content of 5-HTAA in the hippocampal tissue and 202104010910029）；the phosphorylation levels of PI3K and Akt proteins were increased significantly， while the phosphorylation level of NF-κB p65 protein was decreased significantly （P＜0.05）.CONCLUSIONS High-dose of S. italica extract demonstrates significant therapeutic effects on insomnia in mice， and the mechanism of which may be associated with the regulation of PI3K/Akt/NF-κB signaling pathway.

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Drought is a major environmental factor that limits agricultural crop productivity and threatens food security. Foxtail millet is a model crop with excellent abiotic stress tolerance and is consequently an important subject for obtaining a better understanding of the molecular mechanisms underlying plant responses to drought and recovery. Here the physiological and proteomic responses of foxtail millet (cultivar Yugu1) leaves and roots to drought treatments and recovery were evaluated. Drought-treated foxtail millet exhibited increased relative electrolyte leakage and decreased relative water content and chlorophyll content compared to control and rewatering plants. A global analysis of protein profiles was evaluated for drought-treated and recovery treatment leaves and roots. We also identified differentially abundant proteins in drought and recovery groups, enabling comparisons between leaf and root tissue responses to the conditions. The principal component analysis suggested a clear distinction between leaf and root proteomes for the drought-treated and recovery treatment plants. Gene Ontology enrichment and co-expression analyses indicated that the biological responses of leaves differed from those in roots after drought and drought recovery. These results provide new insights and data resources to investigate the molecular basis of tissue-specific functional responses of foxtail millet during drought and recovery, thereby significantly informing crop breeding.

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Foxtail millet (Setaria italica), a vital drought-resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N6 -methyladenosine (m6 A) modification of RNA, a prevalent epi-transcriptomic modification in eukaryotes, provides a binding site for m6 A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521-B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H2 O2 under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m6 A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet. It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m6 A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m6 A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

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Salinity stress is a major threat to crop growth and productivity. Millets are stress-tolerant crops that can withstand the environmental constraints. Foxtail millet is widely recognized as a drought and salinity-tolerant crop owing to its efficient ROS scavenging mechanism. Ascorbate peroxidase (APX) is one of the reactive oxygen species (ROS) scavenging enzymes that leads to hydrogen peroxide (H2O2) detoxification and stabilization of the internal biochemical state of the cell under stress. This inherent capacity of the APX enzyme can further be enhanced by the application of an external mitigant. This study focuses on the impact of salt (NaCl) and selenium (Se) application on the APX enzyme activity of foxtail millet using in silico and in-vitro techniques and mRNA expression studies. The NaCl was applied in the concentrations, i.e., 150 mM and 200 mM, while the Se was applied in 1 µM, 5 µM, and 10 µM concentrations. The in silico studies involved three-dimensional structure modeling and molecular docking. The in vitro studies comprised the morphological and biochemical parameters, alongside mRNA expression studies in foxtail millet under NaCl stress and Se applications. The in silico studies revealed that the APX enzyme showed better interaction with Se as compared to NaCl, thus suggesting the enzyme-modulating role of Se. The morphological and biochemical analysis indicated that Se alleviated the NaCl (150 mM and 200 mM) and induced symptoms at 1 µM as compared to 5 and 10 µM by enhancing the morphological parameters, upregulating the gene expression and enzyme activity of APX, and ultimately reducing the H2O2 content significantly. The transcriptomic studies confirmed the upregulation of chloroplastic APX in response to salt stress and selenium supplementation. Hence, it can be concluded that Se as a mitigant at lower concentrations can alleviate NaCl stress in foxtail millet.

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Dirigent (DIR) proteins play essential roles in regulating plant growth and development, as well as enhancing resistance to abiotic and biotic stresses. However, the whole-genome identification and expression profiling analysis of DIR gene family in millet (Setaria italica (Si)) have not been systematically understood. In this study, we conducted genome-wide identification and expression analysis of the S. italica DIR gene family, including gene structures, conserved domains, evolutionary relationship, chromosomal locations, cis-elements, duplication events, gene collinearity and expression patterns. A total of 38 SiDIR members distributed on nine chromosomes were screened and identified. SiDIR family members in the same group showed higher sequence similarity. The phylogenetic tree divided the SiDIR proteins into six subfamilies: DIR-a, DIR-b/d, DIR-c, DIR-e, DIR-f, and DIR-g. According to the tertiary structure prediction, DIR proteins (like SiDIR7/8/9) themselves may form a trimer to exert function. The result of the syntenic analysis showed that tandem duplication may play the major driving force during the evolution of SiDIRs. RNA-seq data displayed higher expression of 16 SiDIR genes in root tissues, and this implied their potential functions during root development. The results of quantitative real-time PCR (RT-qPCR) assays revealed that SiDIR genes could respond to the stress of CaCl2, CdCl, NaCl, and PEG6000. This research shed light on the functions of SiDIRs in responding to abiotic stress and demonstrated their modulational potential during root development. In addition, the membrane localization of SiDIR7/19/22 was confirmed to be consistent with the forecast. The results above will provide a foundation for further and deeper investigation of DIRs.

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The mitogen-activated protein kinase (MAPK) cascade pathway is a highly conserved plant cell signaling pathway that plays an important role in plant growth and development and stress response. Currently, MAPK cascade genes have been identified and reported in a variety of plants including Arabidopsis thaliana, Oryza sativa, and Triticum aestivum, but have not been identified in foxtail millet (Setaria italica). In this study, a total of 93 MAPK cascade genes, including 15 SiMAPKs, 10 SiMAPKKs and 68 SiMAPKKKs genes, were identified by genome-wide analysis of foxtail millet, and these genes were distributed on nine chromosomes of foxtail millet. Using phylogenetic analysis, we divided the SiMAPKs and SiMAPKKs into four subgroups, respectively, and the SiMAPKKKs into three subgroups (Raf, ZIK, and MEKK). Whole-genome duplication analysis revealed that there are 14 duplication pairs in the MAPK cascade family in foxtail millet, and they are expanded by segmental replication events. Results from quantitative real-time PCR (qRT-PCR) revealed that the expression levels of most SiMAPKs and SiMAPKKs were changed under both exogenous hormone and abiotic stress treatments, with SiMAPK3 and SiMAPKK4-2 being induced under almost all treatments, while the expression of SiMAPKK5 was repressed. In a nutshell, this study will shed some light on the evolution of MAPK cascade genes and the functional mechanisms underlying MAPK cascade genes in response to hormonal and abiotic stress signaling pathways in foxtail millet (Setaria italica).

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Plant malectin/malectin-like receptor-like kinases (MRLKs) play crucial roles throughout the life course of plants. Here, we identified 23 SiMRLK genes from foxtail millet. All the SiMRLK genes were named according to the chromosomal distribution of the SiMRLKs in the foxtail millet genome and grouped into five subfamilies based on phylogenetic relationships and structural features. Synteny analysis indicated that gene duplication events may take part in the evolution of SiMRLK genes in foxtail millet. The expression profiles of 23 SiMRLK genes under abiotic stresses and hormonal applications were evaluated through qRT-PCR. The expression of SiMRLK1, SiMRLK3, SiMRLK7 and SiMRLK19 were significantly affected by drought, salt and cold stresses. Exogenous ABA, SA, GA and MeJA also obviously changed the transcription levels of SiMRLK1, SiMRLK3, SiMRLK7 and SiMRLK19. These results signified that the transcriptional patterns of SiMRLKs showed diversity and complexity in response to abiotic stresses and hormonal applications in foxtail millet.

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Early maturity is an important agronomic trait in most crops, because it can solve the problem of planting in stubble for multiple cropping as well as make full use of light and temperature resources in alpine regions, thereby avoiding damage from low temperatures in the early growth period and early frost damage in the late growth period to improve crop yield and quality. The expression of genes that determine flowering affects flowering time, which directly affects crop maturity and indirectly affects crop yield and quality. Therefore, it is important to analyze the regulatory network of flowering for the cultivation of early-maturing varieties. Foxtail millet (Setaria italica) is a reserve crop for future extreme weather and is also a model crop for functional gene research in C4 crops. However, there are few reports on the molecular mechanism regulating flowering in foxtail millet. A putative candidate gene, SiNF-YC2, was isolated based on quantitative trait loci (QTL) mapping analysis. Bioinformatics analysis showed that SiNF-YC2 has a conserved HAP5 domain, which indicates that it is a member of the NF-YC transcription factor family. The promoter of SiNF-YC2 contains light-response-, hormone-, and stress-resistance-related elements. The expression of SiNF-YC2 was sensitive to the photoperiod and was related to the regulation of biological rhythm. Expression also varied in different tissues and in response to drought and salt stress. In a yeast two-hybrid assay, SiNF-YC2 interacted with SiCO in the nucleus. Functional analysis suggested that SiNF-YC2 promotes flowering and improves resistance to salt stress.

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BACKGROUND: Foxtail millet (Setaria italica) harbors the small diploid genome (~ 450 Mb) and shows the high inbreeding rate and close relationship to several major foods, feed, fuel and bioenergy grasses. Previously, we created a mini foxtail millet, xiaomi, with an Arabidopsis-like life cycle. The de novo assembled genome data with high-quality and an efficient Agrobacterium-mediated genetic transformation system made xiaomi an ideal C4 model system. The mini foxtail millet has been widely shared in the research community and as a result there is a growing need for a user-friendly portal and intuitive interface to perform exploratory analysis of the data. RESULTS: Here, we built a Multi-omics Database for Setaria italica (MDSi, http://sky.sxau.edu.cn/MDSi.htm ), that contains xiaomi genome of 161,844 annotations, 34,436 protein-coding genes and their expression information in 29 different tissues of xiaomi (6) and JG21 (23) samples that can be showed as an Electronic Fluorescent Pictograph (xEFP) in-situ. Moreover, the whole-genome resequencing (WGS) data of 398 germplasms, including 360 foxtail millets and 38 green foxtails and the corresponding metabolic data were available in MDSi. The SNPs and Indels of these germplasms were called in advance and can be searched and compared in an interactive manner. Common tools including BLAST, GBrowse, JBrowse, map viewer, and data downloads were implemented in MDSi. CONCLUSION: The MDSi constructed in this study integrated and visualized data from three levels of genomics, transcriptomics and metabolomics, and also provides information on the variation of hundreds of germplasm resources that can satisfies the mainstream requirements and supports the corresponding research community.

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The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe2+ and Mn2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg2+ uptake in prokaryotes and another the transport of Al3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al3+ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al3+ and Ga3+, which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

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In plants, autophagy plays an important role in regulating intracellular degradation and amino acid recycling in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) shows strong resistance to various abiotic stresses; however, current understanding of the regulation network of abiotic stress resistance in foxtail millet remains limited. In this study, we aimed to determine the autophagy-related gene SiATG8a in foxtail millet. We found that SiATG8a was mainly expressed in the stem and was induced by low-phosphorus (LP) stress. Overexpression of SiATG8a in wheat (Triticum aestivum) significantly increased the grain yield and spike number per m2 under LP treatment compared to those in the WT varieties S366 and S4056. There was no significant difference in the grain P content between SiATG8a-overexpressing wheat and WT wheat under normal phosphorus (NP) and LP treatments. However, the phosphorus (P) content in the roots, stems, and leaves of transgenic plants was significantly higher than that in WT plants under NP and LP conditions. Furthermore, the expression of P transporter genes, such as TaPHR1, TaPHR3, TaIPS1, and TaPT9, in SiATG8a-transgenic wheat was higher than that in WT under LP. Collectively, overexpression of SiATG8a increases the P content of roots, stems, and leaves of transgenic wheat under LP conditions by modulating the expression of P-related transporter gene, which may result in increased grain yield; thus, SiATG8a is a candidate gene for generating transgenic wheat with improved tolerance to LP stress in the field.

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The extension peptide (EP) is the most distinctive feature of mature plant ferritin. Some EPs have exhibited serine-like protease activity, which is associated with iron uptake and release. EP forms a helix and a long loop, followed by a conserved core helical bundle. However, whether the EP adopts a stable or uniform folding pattern in all plants remains unclear. To clarify this, we investigated the crystal structure of ferritin-1 from Setaria italica (SiFer1), a type of monocotyledon. In our structure of SiFer1, the EP is different from other EPs in other solved structures of plant ferritins and consisted of a pair of ß-sheets, a shorter helix, and two loops, which masks two hydrophobic pockets on the outer surface of every subunit. Furthermore, sequence analysis and structure comparison suggest that the EPs in ferritins from monocotyledons may adopt a novel fold pattern, and the conformations of EPs in ferritins are alterable among different plant species. Furthermore, additional eight iron atoms were first founded binding in the fourfold channels, demonstrating the vital function of fourfold channels in iron diffusion. In all, our structure provides new clues for understanding plant ferritins and the functions of the EP.

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BACKGROUND: Foxtail millet (Setaria italica L.) is a millet species with high tolerance to stressful environments. Plant non-specific lipid transfer proteins (nsLTPs) are a kind of small, basic proteins involved in many biological processes. So far, the genome of S. italica has been fully sequenced, and a comprehensive understanding of the evolution and expression of the nsLTP family is still lacking in foxtail millet. RESULTS: Forty-five nsLTP genes were identified in S. italica and clustered into 5 subfamilies except three single genes (SinsLTP38, SinsLTP7, and SinsLTP44). The proportion of SinsLTPs was different in each subfamily, and members within the same subgroup shared conserved exon-intron structures. Besides, 5 SinsLTP duplication events were investigated. Both tandem and segmental duplication contributed to nsLTP expansion in S. italica, and the duplicated SinsLTPs had mainly undergone purifying selection pressure, which suggested that the function of the duplicated SinsLTPs might not diverge much. Moreover, we identified the nsLTP members in 5 other monocots, and 41, 13, 10, 4, and 1 orthologous gene pairs were identified between S. italica and S. viridis, S. bicolor, Z. mays, O. sativa, and B. distachyon, respectively. The functional divergence within the nsLTP orthologous genes might be limited. In addition, the tissue-specific expression patterns of the SinsLTPs were investigated, and the expression profiles of the SinsLTPs in response to abiotic stress were analyzed, all the 10 selected SinsLTPs were responsive to drought, salt, and cold stress. Among the selected SinsLTPs, 2 paired duplicated genes shared almost equivalent expression profiles, suggesting that these duplicated genes might retain some essential functions during subsequent evolution. CONCLUSIONS: The present study provided the first systematic analysis for the phylogenetic classification, conserved domain and gene structure, expansion pattern, and expression profile of the nsLTP family in S. italica. These findings could pave a way for further comparative genomic and evolution analysis of nsLTP family in foxtail millet and related monocots, and lay the foundation for the functional analysis of the nsLTPs in S. italica.

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