Nutrient and moisture limitations reveal keystone metabolites linking rhizosphere metabolomes and microbiomes.

Plants release a wealth of metabolites into the rhizosphere that can shape the composition and activity of microbial communities in response to environmental stress. The connection between rhizodeposition and rhizosphere microbiome succession has been suggested, particularly under environmental stress conditions, yet definitive evidence is scarce. In this study, we investigated the relationship between rhizosphere chemistry, microbiome dynamics, and abiotic stress in the bioenergy crop switchgrass grown in a marginal soil under nutrient-limited, moisture-limited, and nitrogen (N)-replete, phosphorus (P)-replete, and NP-replete conditions. We combined 16S rRNA amplicon sequencing and LC-MS/MS-based metabolomics to link rhizosphere microbial communities and metabolites. We identified significant changes in rhizosphere metabolite profiles in response to abiotic stress and linked them to changes in microbial communities using network analysis. N-limitation amplified the abundance of aromatic acids, pentoses, and their derivatives in the rhizosphere, and their enhanced availability was linked to the abundance of bacterial lineages from Acidobacteria, Verrucomicrobia, Planctomycetes, and Alphaproteobacteria. Conversely, N-amended conditions increased the availability of N-rich rhizosphere compounds, which coincided with proliferation of Actinobacteria. Treatments with contrasting N availability differed greatly in the abundance of potential keystone metabolites; serotonin and ectoine were particularly abundant in N-replete soils, while chlorogenic, cinnamic, and glucuronic acids were enriched in N-limited soils. Serotonin, the keystone metabolite we identified with the largest number of links to microbial taxa, significantly affected root architecture and growth of rhizosphere microorganisms, highlighting its potential to shape microbial community and mediate rhizosphere plant-microbe interactions.

Nutri-cereal tissue-specific transcriptome atlas during development: Functional integration of gene expression to identify mineral uptake pathways in little millet (Panicum sumatrense).

Little millet (Panicum sumatrense Roth ex Roem. & Schult.) is an essential minor millet of southeast Asia and Africa's temperate and subtropical regions. The plant is stress-tolerant, has a short life cycle, and has a mineral-rich nutritional profile associated with unique health benefits. We report the developmental gene expression atlas of little millet (genotype JK-8) from ten tissues representing different stages of its life cycle, starting from seed germination and vegetative growth to panicle maturation. The developmental transcriptome atlas led to the identification of 342 827 transcripts. The BUSCO analysis and comparison with the transcriptomes of related species confirm that this study presents high-quality, in-depth coverage of the little millet transcriptome. In addition, the eFP browser generated here has a user-friendly interface, allowing interactive visualizations of tissue-specific gene expression. Using these data, we identified transcripts, the orthologs of which in Arabidopsis and rice are involved in nutrient acquisition, transport, and response pathways. The comparative analysis of the expression levels of these transcripts holds great potential for enhancing the mineral content in crops, particularly zinc and iron, to address the issue of "hidden hunger" and to attain nutritional security, making it a valuable asset for translational research.

miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress. RESULTS: In total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls. CONCLUSION: Our study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.

Genome-wide analysis of the switchgrass YABBY family and functional characterization of PvYABBY14 in response to ABA and GA stress in Arabidopsis.

BACKGROUND: The small YABBY plant-specific transcription factor has a prominent role in regulating plant growth progress and responding to abiotic stress. RESULTS: Here, a total of 16 PvYABBYs from switchgrass (Panicum virgatum L.) were identified and classified into four distinct subgroups. Proteins within the same subgroup exhibited similar conserved motifs and gene structures. Synteny analyses indicated that segmental duplication contributed to the expansion of the YABBY gene family in switchgrass and that complex duplication events occurred in rice, maize, soybean, and sorghum. Promoter regions of PvYABBY genes contained numerous cis-elements related to stress responsiveness and plant hormones. Expression profile analysis indicated higher expression levels of many PvYABBY genes during inflorescence development and seed maturation, with lower expression levels during root growth. Real-time quantitative PCR analysis demonstrated the sensitivity of multiple YABBY genes to PEG, NaCl, ABA, and GA treatments. The overexpression of PvYABBY14 in Arabidopsis resulted in increased root length after treatment with GA and ABA compared to wild-type plants. CONCLUSIONS: Taken together, our study provides the first genome-wide overview of the YABBY transcription factor family, laying the groundwork for understanding the molecular basis and regulatory mechanisms of PvYABBY14 in response to ABA and GA responses in switchgrass.

Genome-wide association study reveals marker-trait associations for major agronomic traits in proso millet (Panicum miliaceum L.).

MAIN CONCLUSION: The pilot-scale genome-wide association study in the US proso millet identified twenty marker-trait associations for five morpho-agronomic traits identifying genomic regions for future studies (e.g. molecular breeding and map-based cloning). Proso millet (Panicum miliaceum L.) is an ancient grain recognized for its excellent water-use efficiency and short growing season. It is an indispensable part of the winter wheat-based dryland cropping system in the High Plains of the USA. Its grains are endowed with high nutritional and health-promoting properties, making it increasingly popular in the global market for healthy grains. There is a dearth of genomic resources in proso millet for developing molecular tools to complement conventional breeding for developing high-yielding varieties. Genome-wide association study (GWAS) is a widely used method to dissect the genetics of complex traits. In this pilot study of the first-ever GWAS in the US proso millet, 71 globally diverse genotypes of 109 the US proso millet core collection were evaluated for five major morpho-agronomic traits at two locations in western Nebraska, and GWAS was conducted to identify single nucleotide polymorphisms (SNPs) associated with these traits. Analysis of variance showed that there was a significant difference among the genotypes, and all five traits were also found to be highly correlated with each other. Sequence reads from genotyping-by-sequencing (GBS) were used to identify 11,147 high-quality bi-allelic SNPs. Population structure analysis with those SNPs showed stratification within the core collection. The GWAS identified twenty marker-trait associations (MTAs) for the five traits. Twenty-nine putative candidate genes associated with the five traits were also identified. These genomic regions can be used to develop genetic markers for marker-assisted selection in proso millet breeding.

Improvement of little millet (Panicum sumatrense) using novel omics platform and genetic resource integration.

MAIN CONCLUSION: This article explores possible future initiatives, such as the development of targeted breeding and integrated omics approach to boost little millet production, nutritional value, and environmental adaptation. Little millet (P. sumatrense) is a staple grain in many parts of Asia and Africa owing to its abundance in vitamins and minerals and its ability to withstand harsh agro-ecological conditions. Enhancing little millet using natural resources and novel crop improvement strategy is an effective way of boosting nutritional and food security. To understand the genetic makeup of the crop and figure out important characteristics linked to nutritional value, biotic and abiotic resistance, and production, researchers in this field are currently resorting on genomic technology. These realizations have expedited the crop's response to shifting environmental conditions by enabling the production of superior cultivars through targeted breeding. Going forward, further improvements in breeding techniques and genetics may boost the resilience, nutritional content, and production of little millet, which would benefit growers and consumers alike. The research and development on little millet improvement using novel omics platform and the integration of genetic resources are summarized in this review paper. Improved cultivars of little millet that satisfy changing farmer and consumer demands have already been developed through the use of these novel breeding strategies. This article also explores possible future initiatives, such as the development of targeted breeding, genomics, and sustainable agriculture methods. The potential for these measures to boost little millet's overall production, nutritional value, and climate adaptation will be extremely helpful in addressing nutritional security.

Nutritional and nutraceutical richness of neglected little millet genotypes from Eastern Ghats of India: implications for breeding and food value.

MAIN CONCLUSION: The study provides nutritional profiling of unexploited little millets from Eastern Ghats, which has ample opportunities for future breeding programs for enhancing the food quality and holds great potential in food industry. Little millet is an important small millet native to the Indian subcontinent and their nutritional value has been underutilized compared to other cereals. It's nutritional and nutraceutical profiling is essential to integrate the plants in developmental interventions. The present study evaluated comprehensive nutritional, nutraceutical and physico-functional properties of 14 selected little millet genotypes originated from Eastern Ghats of India and compared them with an improved variety (OLM 208) of the locality. The proximate compositions (per 100 g) showed significant variations, with moisture content ranging from 4.13 to 8.48 g, ash from 1.90 to 5.15 g, fat from 2.35 to 5.74 g, protein from 10.46 to 13.83 g, carbohydrate from 70.92 to 77.89 g, fiber from 2.03 to 7.82 g and energy from 372.8 to 391.1 kcal. These little millet flours are rich in phenol 5.37-12.73 mg/g, flavonoid 1.06-8.25 µg/g, vitamin C 12.72-22.86 µg/g, antioxidants 7.22-23.17%, iron 20.38 to 61.60 mg/ kg and zinc 17.47 to 37.59 mg/ kg. The first two components of principal component analysis captures 73.0% of the total variation, which reflected huge variability among the investigated genotypes. Maximum heritability and genetic advance were recorded in flavonoid, fiber, iron, zinc, phenol and vitamin C across the populations. Taken together, some indigenous little millet genotypes such as Mami, Kalia and Bada, were exceptionally rich in fiber, protein, energy, flavonoid, vitamin C and antioxidants and are nutritionally superior compared to other varieties from the locality. These nutrition rich little millet genotypes have ample opportunities for future breeding programs to enhance the cereal quality and holds great potential in food industry for making high value functional foods.

UDP-glycosyltransferase UGT96C10 functions as a novel detoxification factor for conjugating the activated dinitrotoluene sulfonate in switchgrass.

Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water-contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1-hydroxymethyl-2,4-dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O-glucoside as the degradation products of 2,4-DNT-3-SO3Na, the major isoform of DNTS in TNT red water-contaminated soils, in switchgrass via LC-MS/MS- and NMR-based metabolite analyses. Transcriptomic analysis revealed that 15 UDP-glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4-DNT-3-SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4-DNT-3-SO3Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO3Na in liquid medium after 28 days, representing a 3.2-fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10-overexpressing plants may hold great potential for the phytoremediation of TNT red water-contaminated soils.

Genetic linkage map construction and QTL analysis for plant height in proso millet (Panicum miliaceum L.).

KEY MESSAGE: A genetic linkage map representing proso millet genome was constructed with SSR markers, and a major QTL corresponding to plant height was mapped on chromosome 14 of this map. Proso millet (Panicum miliaceum L.) has the lowest water requirements of all cultivated cereal crops. However, the lack of a genetic map and the paucity of genomic resources for this species have limited the utility of proso millet for detailed genetic studies and hampered genetic improvement programs. In this study, 97,317 simple sequence repeat (SSR) markers were developed based on the genome sequence of the proso millet landrace Longmi 4. Using some of these markers in conjunction with previously identified SSRs, an SSR-based linkage map for proso millet was successfully constructed using a large mapping population (316 F2 offspring). In total, 186 SSR markers were assigned to 18 linkage groups corresponding to the haploid chromosomes. The constructed map had a total length of 3033.42 centimorgan (cM) covering 78.17% of the assembled reference genome. The length of the 18 linkage groups ranged from 88.89 cM (Chr. 15) to 274.82 cM (Chr. 16), with an average size of 168.17 cM. To our knowledge, this is the first genetic linkage map for proso millet based on SSR markers. Plant height is one of the most important traits in crop improvement. A major QTL was repeatedly detected in different environments, explaining 8.70-24.50% of the plant height variations. A candidate gene affecting auxin biosynthesis and transport, and ROS homeostasis regulation was predicted. Thus, the linkage map and QTL analysis provided herein will promote the development of gene mining and molecular breeding in proso millet.

Genetically correlated leaf tensile and morphological traits are driven by growing season length in a widespread perennial grass.

PREMISE: Leaf tensile resistance, a leaf's ability to withstand pulling forces, is an important determinant of plant ecological strategies. One potential driver of leaf tensile resistance is growing season length. When growing seasons are long, strong leaves, which often require more time and resources to construct than weak leaves, may be more advantageous than when growing seasons are short. Growing season length and other ecological conditions may also impact the morphological traits that underlie leaf tensile resistance. METHODS: To understand variation in leaf tensile resistance, we measured size-dependent leaf strength and size-independent leaf toughness in diverse genotypes of the widespread perennial grass Panicum virgatum (switchgrass) in a common garden. We then used quantitative genetic approaches to estimate the heritability of leaf tensile resistance and whether there were genetic correlations between leaf tensile resistance and other morphological traits. RESULTS: Leaf tensile resistance was positively associated with aboveground biomass (a proxy for fitness). Moreover, both measures of leaf tensile resistance exhibited high heritability and were positively genetically correlated with leaf lamina thickness and leaf mass per area (LMA). Leaf tensile resistance also increased with the growing season length in the habitat of origin, and this effect was mediated by both LMA and leaf thickness. CONCLUSIONS: Differences in growing season length may promote selection for different leaf lifespans and may explain existing variation in leaf tensile resistance in P. virgatum. In addition, the high heritability of leaf tensile resistance suggests that P. virgatum will be able to respond to climate change as growing seasons lengthen.

Rhizophagus irregularis and biochar can synergistically improve the physiological characteristics of saline-alkali resistance of switchgrass.

Inoculation of arbuscular mycorrhizal fungi (AMF) or biochar (BC) application can improve photosynthesis and promote plant growth under saline-alkali stress. However, little is known about the effects of the two combined on growth and physiological characteristics of switchgrass under saline-alkali stress. This study examined the effects of four treatments: (1) no AMF inoculation and no biochar addition (control), (2) biochar (BC) alone, (3) AMF (Rhizophagus irregularis, Ri) alone, and (4) the combination of both (BC+Ri) on the plant biomass, antioxidant enzymes, chlorophyll, and photosynthetic parameters of switchgrass under saline-alkali stress. The results showed that the above-ground, belowground and total biomass of switchgrass in the BC+Ri treatment group was significantly higher (+136.7%, 120.2% and 132.4%, respectively) than in other treatments compared with Control. BC+Ri treatment significantly increased plant leaves' relative chlorophyll content, antioxidant enzyme activity, and photosynthesis parameters. It is worth noting that the transpiration rate, stomatal conductance, net photosynthetic rate, PSII efficiency and other photosynthetic-related indexes of the BC+Ri treatment group were the highest (38% to 54% higher than other treatments). The fitting results of light response and CO2 response curves showed that the light saturation point, light compensation point, maximum carboxylation rate and maximum electron transfer rate of switchgrass in the Ri+BC treatment group were the highest. In conclusion, biochar combined with Ri has potential beneficial effects on promoting switchgrass growth under saline-alkali stress and improving the activity of antioxidant enzymes and photosynthetic characteristics of plants.

Comparative Multi-Omics Analysis of Broomcorn Millet in Response to Anthracocystis destruens Infection.

Anthracocystis destruens is the causal agent of broomcorn millet (Panicum miliaceum) smut disease, which results in serious yield losses in broomcorn millet production. However, the molecular basis underlying broomcorn millet defense against A. destruens is less understood. In this study, we investigated how broomcorn millet responds to infection by A. destruens by employing a comprehensive multi-omics approach. We examined the responses of broomcorn millet across transcriptome, metabolome, and microbiome levels. Infected leaves exhibited an upregulation of genes related to photosynthesis, accompanied by a higher accumulation of photosynthesis-related compounds and alterations in hormonal levels. However, broomcorn millet genes involved in immune response were downregulated post A. destruens infection, suggesting that A. destruens may suppress broomcorn millet immunity. In addition, we show that the immune suppression and altered host metabolism induced by A. destruens have no significant effect on the microbial community structure of broomcorn millet leaf, thus providing a new perspective for understanding the tripartite interaction between plant, pathogen, and microbiota.

The effect of AMF combined with biochar on plant growth and soil quality under saline-alkali stress: Insights from microbial community analysis.

Arbuscular mycorrhizal fungi (AMF) and biochar application individually can enhance plant tolerance to saline-alkali stress and promote plant growth efficiency. However, little is known about the potential synergistic effects of their combination on improving plant growth and soil quality under saline-alkali stress. This experiment adopted the potted method to explore the effects of four treatments on switchgrass growth and soil quality: biochar (BC), Rhizophagus irregularis (Ri), biochar + Ri (BR) and a control without biochar or Ri (CK). Compared to the CK treatment, the switchgrass biomass increased by 92.4 %, 148.6 %, and 177.3 % in the BC, Ri, and BR treatment groups, respectively. Similarly, the rhizosphere soil quality index increased by 29.33 %, 22.7 %, and 49.1 % in the respective treatment groups. The BR treatment significantly altered the rhizosphere soil microbial composition and diversity. Notably, compared to the other treatments, the archaeal α-diversity in the BR group showed a significant decrease. BR treatment significantly increased the relative abundance of bacteria, fungi and archaea at the genus level (e.g., Bacillus, Trichome and candidatus_methanopenens). Network analysis showed that the complexity and closeness of interactions between different microbial taxa were stronger in the BC, Ri and BR treatments than in the CK treatment, with BR being the more prominent. In summary, biochar combined with Ri has a better effect on promoting the growth of switchgrass under saline-alkali stress, improving the quality of saline-alkali soil, and increasing soil microbial diversity. This study provides a new approach for the efficient development and utilization of saline-alkali land.

Impact of freeze-thaw cycles on greenhouse gas emissions in marginally productive agricultural land under different perennial bioenergy crops.

Knowledge of freeze-thaw-induced carbon (C) and nitrogen (N) cycling and concomitant nitrous oxide (N2O) and carbon dioxide (CO2) emissions in perennial bioenergy crops is crucial to understanding the contribution of these crops in mitigating climate change through reduced greenhouse gas (GHG) emissions. In this study, a 49-day laboratory incubation experiment was conducted to compare the impact of freeze-thaw cycles on N2O and CO2 emissions in different perennial bioenergy crops [miscanthus (Miscanthus giganteus L.), switchgrass (Panicum virgatum L.), and willow (Salix miyabeana L.)] to a successional site and to understand the processes controlling the N2O and CO2 emissions in these crops. The results showed that freeze-thaw cycles caused a decline in dissolved organic C (DOC) and dissolved inorganic N (DIN) concentrations but enhanced the dissolved organic N (DON) and nitrate (NO3-). Although, freeze-thaw decreased water stable soil aggregates in all the bioenergy crops and successional site, this did not have any significant impact on N2O and CO2 emissions, suggesting that the N2O and CO2 emitted during the freeze-thaw cycles may have originated mostly from cellular materials released from lysis and death of microbial biomass rather than from soil aggregate disruption. Cumulative N2O emissions measured over the 49-day incubation period ranged from 148 mg N2O-N m-2 to 17 mg N2O-N m-2 and were highest in miscanthus followed by willow, switchgrass, and successional site. Cumulative CO2 on the other hand was highest in the successional site than any of the bioenergy crops and ranged from 25,262 mg CO2-C m-2 to 15,403 mg CO2-C m-2 after the 49 days. Higher N2O emissions in the miscanthus and willow than switchgrass and successional site were attributed to accelerated N losses as N2O. Results from our study indicate that managing perennial bioenergy crops on low productive agricultural lands to reduce freeze-thaw related GHG emissions and climate change mitigation is dependent on the crop species grown.

Application of CRISPR/Cas12i.3 for targeted mutagenesis in broomcorn millet (Panicum miliaceum L.).

A CRISPR/Cas12i.3-based gene editing platform is established in broomcorn millet (Panicum miliaceum) and used to create new elite germplasm for this ancient crop.

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

Enhancement of the germination and growth of Panicum miliaceum and Brassica juncea in Cd- and Zn-contaminated soil inoculated with heavy-metal-tolerant Leifsonia sp. ZP3.

The addition of plant-growth-promoting bacteria (PGPB) to heavy-metal-contaminated soils can significantly improve plant growth and productivity. This study isolated heavy-metal-tolerant bacteria with growth-promoting traits and investigated their inoculation effects on the germination rates and growth of millet (Panicum miliaceum) and mustard (Brassica juncea) in Cd- and Zn-contaminated soil. Leifsonia sp. ZP3, which is resistant to Cd (0.5 mM) and Zn (1 mM), was isolated from forest soil. The ZP3 strain exhibited plant-growth-promoting activity, including indole-3-acetic acid production, phosphate solubilization, catalase activity, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging. In soil contaminated with low concentrations of Cd (0.232 ± 0.006 mM) and Zn (6.376 ± 0.256 mM), ZP3 inoculation significantly increased the germination rates of millet and mustard 8.35- and 31.60-fold, respectively, compared to the non-inoculated control group, while the shoot and root lengths of millet increased 1.77- and 4.44-fold (p < 0.05). The chlorophyll content and seedling vigor index were also 4.40 and 18.78 times higher in the ZP3-treated group than in the control group (p < 0.05). The shoot length of mustard increased 1.89-fold, and the seedling vigor index improved 53.11-fold with the addition of ZP3 to the contaminated soil (p < 0.05). In soil contaminated with high concentrations of Cd and Zn (0.327 ± 0.016 and 8.448 ± 0.250 mM, respectively), ZP3 inoculation led to a 1.98-fold increase in the shoot length and a 2.07-fold improvement in the seedling vigor index compared to the control (p < 0.05). The heavy-metal-tolerant bacterium ZP3 isolated in this study thus represents a promising microbial resource for improving the efficiency of phytoremediation in Cd- and Zn-contaminated soil.

Genome-wide identification of GRF transcription factors and their expression profile in stem meristem of broomcorn millet (Panicum miliaceum L.).

To understand the genome-wide information of the GRF family genes in broomcorn millet and their expression profile in the vegetative meristems, bioinformatic methods and transcriptome sequencing were used to analyze the characteristics, physical and chemical properties, phylogenetic relationship, chromosome distribution, gene structure, cis-acting elements and expression profile in stem meristem for the GRF family members. The results showed that the GRF gene family of millet contains 21 members, and the PmGRF gene is unevenly distributed on 12 chromosomes. The lengths of PmGRF proteins vary from 224 to 618 amino acids, and the isoelectric points are between 4.93-9.69. Each member of the family has 1-4 introns and 2-5 exons. The protein PmGRF13 is localized in both the nucleus and chloroplast, and the rest PmGRF proteins are located in the nucleus. Phylogenetic analysis showed that the 21 GRF genes were divided into 4 subfamilies (A,B,C and D) in broomcorn millet. The analysis of cis-acting elements showed that there were many cis-acting elements involved in light response, hormone response, drought induction, low temperature response and other environmental stress responses in the 2000 bp sequence upstream of the GRF genes. Transcriptome sequencing and qRT-PCR analyses showed that the expression levels of PmGRF3 and PmGRF12 in the dwarf variety Zhang778 were significantly higher than those of the tall variety Longmi12 in the internode and node meristems at the jointing stage, while the expression patterns of PmGRF4, PmGRF16 and PmGRF21 were reverse. In addition, the expression levels of PmGRF2 and PmGRF5 in the internode of Zhang778 were significantly higher than Longmi12. The other GRF genes were not or insignificantly expressed. These results indicated that seven genes, PmGRF2, PmGRF3, PmGRF4, PmGRF5, PmGRF12, PmGRF16 and PmGRF21, were related to the formation of plant height in broomcorn millet.

A complete reference genome of broomcorn millet.

Broomcorn millet (Panicum miliaceum L.), known for its traits of drought resistance, adaptability to poor soil, short growth period, and high photosynthetic efficiency as a C4 plant, represents one of the earliest domesticated crops globally. This study reports the telomere-to-telomere (T2T) gap-free reference genome for broomcorn millet (AJ8) using PacBio high-fidelity (HiFi) long reads, Oxford Nanopore long-read technologies and high-throughput chromosome conformation capture (Hi-C) sequencing data. The size of AJ8 genome was approximately 834.7 Mb, anchored onto 18 pseudo-chromosomes. Notably, 18 centromeres and 36 telomeres were obtained. The assembled genome showed high quality in terms of completeness (BUSCO score: 99.6%, QV: 61.7, LAI value: 20.4). In addition, 63,678 protein-coding genes and 433.8 Mb (~52.0%) repetitive sequences were identified. The complete reference genome for broomcorn millet provides a valuable resource for genetic studies and breeding of this important cereal crop.