Understanding the responses of tillering to 2,4-D isooctyl ester in Setaria viridis L.

BACKGROUND: Green foxtail [Setaria viridis (L.)] is one of the most abundant and troublesome annual grass weeds in alfalfa fields in Northeast China. Synthetic auxin herbicide is widely used in agriculture, while how auxin herbicide affects tillering on perennial grass weeds is still unclear. A greenhouse experiment was conducted to examine the effects of auxin herbicide 2,4-D on green foxtail growth, especially on tillers. RESULTS: In the study, 2,4-D isooctyl ester was used. There was an inhibition of plant height and fresh weight on green foxtail after application. The photosynthetic rate of the leaves was dramatically reduced and there was an accumulation of malondialdehyde (MDA) content. Moreover, applying 2,4-D isooctyl ester significantly reduced the tillering buds at rates between 2100 and 8400 ga. i. /ha. Transcriptome results showed that applying 2,4-D isooctyl ester on leaves affected the phytohormone signal transduction pathways in plant tillers. Among them, there were significant effects on auxin, cytokinin, abscisic acid (ABA), gibberellin (GA), and brassinosteroid signaling. Indeed, external ABA and GA on leaves also limited tillering in green foxtail. CONCLUSIONS: These data will be helpful to further understand the responses of green foxtail to 2, 4-D isooctyl ester, which may provide a unique perspective for the development and identification of new target compounds that are effective against this weed species.

Transcriptional and metabolic profiling of sulfur starvation response in two monocots.

BACKGROUND: Sulfur (S) is a mineral nutrient essential for plant growth and development, which is incorporated into diverse molecules fundamental for primary and secondary metabolism, plant defense, signaling, and maintaining cellular homeostasis. Although, S starvation response is well documented in the dicot model Arabidopsis thaliana, it is not clear if the same transcriptional networks control the response also in the monocots. RESULTS: We performed series of physiological, expression, and metabolite analyses in two model monocot species, one representing the C3 plants, Oryza sativa cv. kitaake, and second representing the C4 plants, Setaria viridis. Our comprehensive transcriptomic analysis revealed twice as many differentially expressed genes (DEGs) in S. viridis than in O. sativa under S-deficiency, consistent with a greater loss of sulfur and S-containing metabolites under these conditions. Surprisingly, most of the DEGs and enriched gene ontology terms were species-specific, with an intersect of only 58 common DEGs. The transcriptional networks were different in roots and shoots of both species, in particular no genes were down-regulated by S-deficiency in the roots of both species. CONCLUSIONS: Our analysis shows that S-deficiency seems to have different physiological consequences in the two monocot species and their nutrient homeostasis might be under distinct control mechanisms.

Assembly and comparative analysis of the first complete mitochondrial genome of Setaria italica.

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.

Genome-wide identification of the MED25 BINDING RING-H2 PROTEIN gene family in foxtail millet (Setaria italica L.) and the role of SiMBR2 in resistance to abiotic stress in Arabidopsis.

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.

Identifying the mechanistic basis to nitrogen responsiveness in two contrasting Setaria italica accessions.

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 remobilising flag leaf N compared to 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 are potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

Systematic Analysis of DNA Demethylase Gene Families in Foxtail Millet (Setaria italica L.) and Their Expression Variations after Abiotic Stresses.

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.

Physiological Investigation and Transcriptome Analysis Reveals the Mechanisms of Setaria italica's Yield Formation under Heat Stress.

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.

Genome-wide loss of CHH methylation with limited transcriptome changes in Setaria viridis DOMAINS REARRANGED METHYLTRANSFERASE (DRM) mutants.

The DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole-genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for the locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild-type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.

Elucidating the role of SWEET13 in phloem loading of the C4 grass Setaria viridis.

Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.

SvSTL1 in the large subunit family of ribonucleotide reductases plays a major role in chloroplast development of Setaria viridis.

Ribonucleotide reductases (RNRs) are essential enzymes in DNA synthesis. However, little is known about the RNRs in plants. Here, we identified a svstl1 mutant from the self-created ethyl methanesulfonate (EMS) mutant library of Setaria viridis. The mutant leaves exhibited a bleaching phenotype at the heading stage. Paraffin section analysis showed the destruction of the C4 Kranz anatomy. Transmission electron microscopy results further demonstrated the severely disturbed development of some chloroplasts. MutMap analysis revealed that the SvSTL1 gene is the primary candidate, encoding a large subunit of RNRs. Complementation experiments confirmed that SvSTL1 is responsible for the phenotype of svstl1. There are two additional RNR large subunit homologs in S. viridis, SvSTL2 and SvSTL3. To further understand the functions of these three RNR large subunit genes, a series of mutants were generated via CRISPR/Cas9 technology. In striking contrast to the finding that all three SvSTLs interact with the RNR small subunit, the phenotype varied along with the copies of chloroplast genome among different svstl single mutants: the svstl1 mutant exhibited pronounced chloroplast development and significantly fewer copies of the chloroplast genome than the svstl2 or svstl3 single mutants. These results suggested that SvSTL1 plays a major role in the optimal function of RNRs and is essential for chloroplast development. Furthermore, through the analysis of double and triple mutants, the study provides new insights into the finely tuned coordination among SvSTLs to maintain normal chloroplast development in the emerging C4 model plant S. viridis.

Overexpression of a Plant-Specific Gγ Protein, AGG3, in the Model Monocot Setaria viridis Confers Tolerance to Heat Stress.

The vascular plant-specific, cysteine-rich type III Gγ proteins, which are integral components of the heterotrimeric G-protein complex, play crucial roles in regulating a multitude of plant processes, including those related to crop yield and responses to abiotic stresses. The presence of multiple copies of type III Gγ proteins in most plants and a propensity of the presence of specific truncated alleles in many cultivated crops present an ambiguous picture of their roles in modulating specific responses. AGG3 is a canonical type III Gγ protein of Arabidopsis, and its overexpression in additional model crops offers the opportunity to directly evaluate the effects of protein expression levels on plant phenotypes. We have shown that AGG3 overexpression in the monocot model Setaria viridis leads to an increase in seed yield. In this study, we have investigated the response of the S. viridis plants overexpressing AGG3 to heat stress (HS), one of the most important abiotic stresses affecting crops worldwide. We show that a short span of HS at a crucial developmental time point has a significant effect on plant yield in the later stages. We also show that plants with higher levels of AGG3 are more tolerant to HS. This is attributed to an altered regulation of stress-responsive genes and improved modulation of the photosynthetic efficiency during the stress. Overall, our results confirm that AGG3 plays a crucial role in regulating plant responses to unfavorable environmental conditions and may contribute positively to avoiding crop yield losses.

MDSi: Multi-omics Database for Setaria italica.

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.

Genome-wide identification and expression analysis of the SAUR gene family in foxtail millet (Setaria italica L.).

BACKGROUND: Auxin performs important functions in plant growth and development processes, as well as abiotic stress. Small auxin-up RNA (SAUR) is the largest gene family of auxin-responsive factors. However, the knowledge of the SAUR gene family in foxtail millet is largely obscure. RESULTS: In the current study, 72 SiSAUR genes were identified and renamed according to their chromosomal distribution in the foxtail millet genome. These SiSAUR genes were unevenly distributed on nine chromosomes and were classified into three groups through phylogenetic tree analysis. Most of the SiSAUR members from the same group showed similar gene structure and motif composition characteristics. Analysis of cis-acting elements showed that many hormone and stress response elements were identified in the promoter region of SiSAURs. Gene replication analysis revealed that many SiSAUR genes were derived from gene duplication events. We also found that the expression of 10 SiSAURs was induced by abiotic stress and exogenous hormones, which indicated that SiSAUR genes may participated in complex physiological processes. CONCLUSIONS: Overall, these results will be valuable for further studies on the biological role of SAUR genes in foxtail development and response to stress conditions and may shed light on the improvement of the genetic breeding of foxtail millet.

The YABBY gene SHATTERING1 controls activation rather than patterning of the abscission zone in Setaria viridis.

Abscission is predetermined in specialized cell layers called the abscission zone (AZ) and activated by developmental or environmental signals. In the grass family, most identified AZ genes regulate AZ anatomy, which differs among lineages. A YABBY transcription factor, SHATTERING1 (SH1), is a domestication gene regulating abscission in multiple cereals, including rice and Setaria. In rice, SH1 inhibits lignification specifically in the AZ. However, the AZ of Setaria is nonlignified throughout, raising the question of how SH1 functions in species without lignification. Crispr-Cas9 knockout mutants of SH1 were generated in Setaria viridis and characterized with histology, cell wall and auxin immunofluorescence, transmission electron microscopy, hormonal treatment and RNA-Seq analysis. The sh1 mutant lacks shattering, as expected. No differences in cell anatomy or cell wall components including lignin were observed between sh1 and the wild-type (WT) until abscission occurs. Chloroplasts degenerated in the AZ of WT before abscission, but degeneration was suppressed by auxin treatment. Auxin distribution and expression of auxin-related genes differed between WT and sh1, with the signal of an antibody to auxin detected in the sh1 chloroplast. SH1 in Setaria is required for activation of abscission through auxin signaling, which is not reported in other grass species.

The role of SWEET4 proteins in the post-phloem sugar transport pathway of Setaria viridis sink tissues.

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed.

Unique features of regulation of sulfate assimilation in monocots.

Sulfate assimilation is an essential pathway of plant primary metabolism, regulated by the demand for reduced sulfur (S). The S-containing tripeptide glutathione (GSH) is the key signal for such regulation in Arabidopsis, but little is known about the conservation of these regulatory mechanisms beyond this model species. Using two model monocot species, C3 rice (Oryza sativa) and C4Setaria viridis, and feeding of cysteine or GSH, we aimed to find out how conserved are the regulatory mechanisms described for Arabidopsis in these species. We showed that while in principle the regulation is similar, there are many species-specific differences. For example, thiols supplied by the roots are translocated to the shoots in rice but remain in the roots of Setaria. Cysteine and GSH concentrations are highly correlated in Setaria, but not in rice. In both rice and Setaria, GSH seems to be the signal for demand-driven regulation of sulfate assimilation. Unexpectedly, we observed cysteine oxidation to sulfate in both species, a reaction that does not occur in Arabidopsis. This reaction is dependent on sulfite oxidase, but the enzyme(s) releasing sulfite from cysteine still need to be identified. Altogether our data reveal a number of unique features in the regulation of S metabolism in the monocot species and indicate the need for using multiple taxonomically distinct models to better understand the control of nutrient homeostasis, which is important for generating low-input crop varieties.

Molecular evolution, diversification, and expression assessment of MADS gene family in Setaria italica, Setaria viridis, and Panicum virgatum.

KEY MESSAGE: This paper sheds light on the evolution and expression patterns of MADS genes in Setaria and Panicum virgatum. SiMADS51 and SiMADS64 maybe involved in the ABA-dependent pathway of drought response. The MADS gene family is a key regulatory factor family that controls growth, reproduction, and response to abiotic stress in plants. However, the molecular evolution of this family is rarely reported. Here, a total of 265 MADS genes were identified in Setaria italica (foxtail millet), Setaria viridis (green millet), and Panicum virgatum (switchgrass) and analyzed by bioinformatics, including physicochemical characteristics, subcellular localization, chromosomal position and duplicate, motif distribution, genetic structure, genetic evolvement, and expression patterns. Phylogenetic analysis was used to categorize these genes into M and MIKC types. The distribution of motifs and gene structure were similar for the corresponding types. According to a collinearity study, the MADS genes have been mostly conserved during evolution. The principal cause of their expansion is segmental duplication. However, the MADS gene family tends to shrink in foxtail millet, green millet, and switchgrass. The MADS genes were subjected to purifying selection, but several positive selection sites were also identified in three species. And most of the promoters of MADS genes contain cis-elements related to stress and hormonal response. RNA-seq and quantitative Real-time PCR (qRT-PCR) analysis also were examined. SiMADS genes expression levels are considerably changed in reaction to various treatments, following qRT-PCR analysis. This sheds fresh light on the evolution and expansion of the MADS family in foxtail millet, green millet, and switchgrass, and lays the foundation for further research on their functions.

First Report of Bipolaris zeae Causing Leaf Spot on Setaria viridis in China.

Green bristlegrass (Setaria viridis) is an annual dominant herb in arid sandy grasslands with strong drought resistance to water stress (Valença et al. 2020). In August 2021, brown or dark tan spots on the leaves of S. viridis were observed on natural grassland in Qingyang city, Gansu Province, China (36.55°N, 107.32°E). Leaf disease incidence was around 13%, and the infected area of each leaf was about 20%. For isolation, 30 tissue pieces (5×5 mm) from 10 symptomatic leaf samples were surface-sterilized with 70% ethanol for 30 seconds and rinsed three times with steriled distilled water. Then these tissues were placed on the potato dextrose agar (PDA) at 25 °C, and incubated in dark from 2 to 5 days. Sixteen single-spored cultures with consistent colony characteristics were obtained by single spore isolation as described in Manamgoda et al. (2012). Colonies on PDA were irregular shapes with black color and white edge; conidia were dark brown with septate, cylindrical, straight or flexuous geniculate at the upper end, 22.7 to 69.38×9.2 to 17.6 µm (n = 50; average size 40.8×13.2 µm) under microscope (40X). The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions of the representative isolate 83HXGWC, 84HXGWC and 85HXGWC were amplified using the primers of ITS1/ITS4 and GPD1/GPD2 (Manamgoda et al. 2012). All the amplified gene sequences were deposited into GenBank with accession numbers ITS (OP703331, OQ978850, and OQ978851) and GPD (OQ559683, OQ992505, and OQ992506). BLAST analysis of both the two segments showed 100% identity with those reported sequences of ex-type isolate BRIP 11512 (KJ415538 and KJ415408) of B. zeae Sivan (Tan et al. 2014; Bhunjun et al. 2020), respectively. In the maximum-likelihood phylogenetic tree, 83-HXGWC, 84HXGWC and 85HXGWC strains were grouped with the reference sequences of B. zeae with a high bootstrap supporting values of 100%. Five healthy green bristlegrass (6 weeks) plants in pots were sprayed with conidial suspensions (1×106 conidia ml-1). Another five pots were sprayed with sterilized distilled water (controls). Then all pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse maintained at 18 to 25 °C. After incubation for 14 days, the typical symptoms of leaf spots developed from brown to dark brown on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The measurement of pathogenicity was carried out three times. The same pathogens were consistently reisolated from inoculated leaves, and was confirmed as B. zeae based on morphological and molecular analyses, fulfilling Koch's postulates. This study provides a new record of B. zeae on green bristlegrass on natural grassland in China, which can potentially impose disease challenges to other crops. Since, B. oryzae has been known to cause disease in cereal crops (Manamgoda et al. 2014), this can be a potential alternate source of survival of this pathogen.

SiNF-YC2 Regulates Early Maturity and Salt Tolerance in Setaria italica.

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.

The m6 A reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica.

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.