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The two-component system (TCS) consists of histidine kinase (HK), histidine phosphate transfer protein (HP), and response regulatory factor (RR). It is one of the most crucial components of signal transduction in plants, playing a significant role in regulating plant growth, development, and responses to various abiotic stresses. Although TCS genes have been extensively identified in a variety of plants, the genome-wide recognition and examination of TCS in switchgrass remain unreported. Accordingly, this study identified a total of 87 TCS members in the genome of switchgrass, comprising 20 HK(L)s, 10 HPs, and 57 RRs. Detailed analyses were also conducted on their gene structures, conserved domains, and phylogenetic relationships. Moreover, this study analysed the gene expression profiles across diverse organs and investigated their response patterns to adverse environmental stresses. Results revealed that 87 TCS genes were distributed across 18 chromosomes, with uneven distribution. Expansion of these genes in switchgrass was achieved through both fragment and tandem duplication. PvTCS members are relatively conservative in the evolutionary process, but the gene structure varies significantly. Various cis-acting elements, varying in types and amounts, are present in the promoter region of PvTCSs, all related to plant growth, development, and abiotic stress, due to the TCS gene structure. Protein-protein interaction and microRNA prediction suggest complex interactions and transcriptional regulation among TCS members. Additionally, most TCS members are expressed in roots and stems, with some genes showing organ-specific expression at different stages of leaf and inflorescence development. Under conditions of abiotic stress such as drought, low temperature, high temperature, and salt stress, as well as exogenous abscisic acid (ABA), the expression of most TCS genes is either stimulated or inhibited. Our systematic analysis could offer insight into the characterization of the TCS genes, and further the growth of functional studies in switchgrass., (© 2024. The Author(s).)

Soil alkalization has become a serious problem that limits plant growth through osmotic stress, ionic imbalance, and oxidative stress. Understanding how plants resist alkali stress has practical implications for alkaline-land utilization. In this study, we identified a small GTPase, PvARFR2 (ADP ribosylation factors related 2), that positively regulates alkali tolerance in switchgrass (Panicum virgatum) and uncovered its potential mode of action. Overexpressing PvARFR2 in switchgrass and Arabidopsis (Arabidopsis thaliana) conferred transformant tolerance to alkali stress, demonstrated by alleviated leaf wilting, less oxidative injury, and a lower Na+/K+ ratio under alkali conditions. Conversely, switchgrass PvARFR2-RNAi and its homolog mutant atgb1 in Arabidopsis displayed alkali sensitives. Transcriptome sequencing analysis showed that cytosolic abscisic acid (ABA) receptor kinase PvCARK3 transcript levels were higher in PvARFR2 overexpression lines compared to the controls and were strongly induced by alkali treatment in shoots and roots. Phenotyping analysis revealed that PvCARK3-OE × atgb1 lines were sensitive to alkali similar to the Arabidopsis atgb1 mutant, indicating that PvARFR2/AtGB1 functions in the same pathway as PvCARK3 under alkaline stress conditions. Application of ABA on PvARFR2-OE and PvCARK3-OE switchgrass transformants resulted in ABA sensitivity. Moreover, we determined that PvARFR2 physically interacts with PvCARK3 in vitro and in vivo. Our results indicate that a small GTPase, PvARFR2, positively responds to alkali stress by interacting with the cytosolic ABA receptor kinase PvCARK3, connecting the alkaline stress response to ABA signaling., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

To elucidate the mechanisms underlying photoperiodic responses, we investigated the genomic and metabolomic responses of two broomcorn millet (Panicum miliaceum L.) genotypes. For this purpose, light-insensitive (D32) and light-sensitive (M51) genotypes were exposed to a 16 h photoperiod (long-day (LD) conditions) and an 8 h photoperiod (short-day (SD) conditions), and various transcriptomic and metabolomic changes were investigated. A total of 1664, 2564, 13,017, and 15548 DEGs were identified in the SD-D, LD-D, LD-M, and SD-M groups, respectively. Furthermore, 112 common DEGs were identified as well. Interestingly, most DEGs in the different groups were associated with photosynthesis and phenylpropanoid and carotenoid biosynthesis. In addition, 822 metabolites were identified under different treatments. The main metabolites, including L-malic and fumaric acids, were identified in the negative mode, whereas brucine and loperamide were identified in the positive mode. KEGG analysis revealed that the metabolites in the different groups were enriched in the same metabolic pathway of the TCA cycle. Furthermore, in negative mode, the metabolites of M51 were mainly D-glucose, whereas those of D32 were mainly L-malic and fumaric acids. One photoperiod candidate gene (C2845_PM11G01290), annotated as ATP6B, significantly increased the levels of L-malic and fumaric acids. In conclusion, our study provides a theoretical basis for understanding the molecular mechanisms of photoperiodic response regulation and can be used as a reference for marker development and resource identification in Panicum miliaceum L.., (© 2024. The Author(s).)

The heavy metal cadmium (Cd), known for its high toxicity, poses a grave threat to human health through the food chain. N 6 -methyladenosine (m 6 A), the most abundant internal modification, regulates plant adaptation to various adversities, yet the panorama of m 6 A modifications in switchgrass under cadmium stress remains elusive. This study examines the physiological responses of switchgrass roots and shoots exposed to 50 μM CdCl 2 , alongside an overview of transcriptome-wide m 6 A methylation patterns. After cadmium treatment, methylation modifications are primarily enriched near stop codons and the 3'UTR region, with a negative correlation between m 6 A modification and gene expression levels. In shoots, approximately 58 % of DEGs with m 6 A modifications show upregulation in expression and decrease in m 6 A peaks, including zinc transporter 4-like (ZIP4). In roots, about 43 % of DEGs with m 6 A modifications exhibit downregulation in expression and increase in m 6 A peaks, such as the ABC transporter family member (ABCG25). We further validate the m 6 A enrichment, gene expression and mRNA stability of ZIP4 in response to Cd treatment. The results suggest that the negative correlation of m 6 A enrichment and gene expression is due to altered mRNA stability. Our study establishes an m 6 A regulatory network governing cadmium transport in switchgrass roots and shoots, offering new avenues for candidate gene manipulation in phytoremediation applications of heavy metal pollution., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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-SO 3 Na, 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-SO 3 Na 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-SO 3 Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO 3 Na 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., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73 g/L in shake flask and 15.71 ± 0.80 g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2024 His Majesty the King in Right of Canada and The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. Reproduced with the permission of the Minister of Agriculture and Agri‐Food Canada.)

N 6 -methyladenosine (m 6 A), a nucleotide modification that is frequently seen in RNA, plays a crucial role in plant growth, development and stress resistance. However, the m 6 A regulatory machinery in switchgrass (Panicum virgatum L.), a model plant for cellulose-to-ethanol conversion, remains largely unknown. In this study, we identified 57 candidate genes involved in m 6 A-regulation in the switchgrass genome, and analyzed their chromosomal distribution, evolutionary relationships, and functions. Notably, we observed distinct gene expression patterns under salt and drought stress, with salt stress inducing writer and eraser genes, alongside drought stress predominantly affecting reader genes. Additionally, we knocked out PvALKBH10, an m 6 A demethylase gene, via CRISPR/Cas9 and found its potential function in controlling flowering time. This study provides insight into the genomic organization and evolutionary features of m 6 A-associated putative genes in switchgrass, and therefore serves as the basis for further functional studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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 (miR156 OE ) 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 miR156 OE 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 miR156 OE 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., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

Applying extracts from plants is considered a safe approach in biomedicine and bio-nanotechnology. The present report is considered the first study that evaluated the seeds of Lasiurus scindicus and Panicum turgidum as biogenic agents in the synthesis of silver nanoparticles (AgNPs) which had bioactivity against cancer cells and bacteria. Assessment of NPs activity against varied cell lines (colorectal cancer HCT116 and breast cancer MDA MBA 231 and MCF 10A used as control) was performed beside the antibacterial efficiency. Different techniques (DLS, TEM, EDX and FTIR) were applied to characterize the biosynthesized AgNPs. The phytochemicals from both L. scindicus and Panicum turgidum were identified by GC-MS analysis. Spherical monodisperse NPs at average diameters of 149.6 and 100.4 nm were obtained from seed extract of L. scindicus (L-AgNPs) and P. turgidum, (P-AgNPs) respectively. A strong absorption peak at 3 keV is observed by the EDX spectrum in the tested NPs. Our study provided effective NPs in mitigating the tested cell lines and the lowest IC 50 were 7.8 and 10.30 for MDA MB231 treated by L-AgNPs and P-AgNPs, respectively. Both fabricated NPs might differentially target the MDA MB231 cells compared to HCT116 and MCF10A. Ultrastructural changes and damage for the NPs-treated MDA MB231 cells were studied using TEM and LSM analysis. Antibacterial activity was also observed. About 200 compounds were identified in L. scindicus and P. turgidum by GC-MS analysis might be responsible for the NPs reduction and capping abilities. Efficient NPs against cancer cells and microbes were obtained, however large-scale screening is needed to validate our findings., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Switchgrass (Panicum virgatum L.) plays a pivotal role as a bioenergy feedstock in the production of cellulosic ethanol and contributes significantly to enhancing ecological grasslands and soil quality. The utilization of non-coding RNAs (ncRNAs) has gained momentum in deciphering the intricate genetic responses to abiotic stress in various plant species. Nevertheless, the current research landscape lacks a comprehensive exploration of the responses of diverse ncRNAs, including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), to drought stress in switchgrass. In this study, we employed whole transcriptome sequencing to comprehensively characterize the expression profiles of both mRNA and ncRNAs during episodes of drought stress in switchgrass. Our analysis identified a total of 12,511 mRNAs, 59 miRNAs, 38 circRNAs, and 368 lncRNAs that exhibited significant differential expression between normal and drought-treated switchgrass leaves. Notably, the majority of up-regulated mRNAs displayed pronounced enrichment within the starch and sucrose metabolism pathway, as validated through KEGG analysis. Co-expression analysis illuminated that differentially expressed (DE) lncRNAs conceivably regulated 1308 protein-coding genes in trans and 7110 protein-coding genes in cis. Furthermore, both cis- and trans-target mRNAs of DE lncRNAs exhibited enrichment in four common KEGG pathways. The intricate interplay between lncRNAs and circRNAs with miRNAs via miRNA response elements was explored within the competitive endogenous RNA (ceRNA) network framework. As a result, we constructed elaborate regulatory networks, including lncRNA-novel&#95;miRNA480-mRNA, lncRNA-novel&#95;miRNA304-mRNA, lncRNA/circRNA-novel&#95;miRNA122-PvSS4, and lncRNA/circRNA-novel&#95;miRNA14-PvSS4, and subsequently validated the functionality of the target gene, starch synthase 4 (PvSS4). Furthermore, through the overexpression of PvSS4, we ascertained its capacity to enhance drought tolerance in yeast. However, it is noteworthy that PvSS4 did not exhibit any discernible impact under salt stress conditions. These findings, as presented herein, not only contribute substantively to our understanding of ceRNA networks but also offer a basis for further investigations into their potential functions in response to drought stress in switchgrass., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Recent findings suggest that optimal application of nitrogen fertilizers can effectively improve the quality of proso millet (PM). Here, we aimed to investigate the pathways associated with starch synthesis and metabolism to elucidate the effect and molecular mechanisms of nitrogen fertilization in starch synthesis and properties in waxy and non-waxy PM varieties using transcriptomic techniques. Co-expression network analysis revealed that the regulation of starch synthesis and quality in PM by nitrogen fertilizer mainly occurred in the S2 and S3 stages during grain filling. Nitrogen fertilization inhibited glycolysis/gluconeogenesis and starch biosynthesis in grains, but increased starch degradation to maltose and dextrin and then to glucose. Moreover, nitrogen fertilization increased starch accumulation by upregulating the expression of SuS and malZ genes, thereby increasing the total starch content in grains. In contrast, nitrogen fertilization suppressed the expression of GBSS gene and decreased amylose content in PM grains, resulting in a relatively higher crystallinity, light transmittance, and breakdown viscosity in the two PM varieties. Overall, these results provided transcriptomics insights into the molecular mechanisms by which nitrogen fertilization regulates starch quality in PM, identified key genes that associated with the starch properties, and provided new insights into the quality cultivation of PM., Competing Interests: Declaration of competing interest There are no conflicts of interest regarding this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Polycyclic aromatic hydrocarbons (PAHs), as priority organic pollutants, are capable of accumulation in plants. Phenanthrene (Phe) is one of the most abundant low-molecular-weight PAH in the environment which is commonly used as a model PAH in many phytoremediation studies and as a representative compound for all PAHs group. This paper highlights the uptake, translocation, and accumulation of Phe by growing proso millet (Panicum miliaceum L.) in a pot experiment, subjected to 500, 1000, 1500, and 2000 ppm of Phe treatment after 15 and 30 days. Phe naturally existed in P. miliaceum and its concentration showed a time-dependent reduction in treated plant tissues as well as in perlites. Phe concentration in shoots was higher than in roots. During the aging process, the uptake of Phe was diminished whereas translocation factor (TF) demonstrated an overall increasing trend among treatments. The shoot concentration factor (SCF) values were higher than those of root concentration factor (RCF) on both days 15 and 30 and the highest values for both parameters were achieved in 500 ppm of Phe. Both RCFs and SCFs generally tended to decrease with the increase of perlite Phe concentrations. These results suggested that Phe tended to transfer to the shoots and be metabolized there. The Phe concentration revealed a significant decline in all levels of treatment on both 15 (84 to 96%) and 30 (76 to 94%) days. Therefore, the presence of P. miliaceum was effective in promoting the phytoremediation of Phe polluted perlites., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Switchgrass is one of the most promising bioenergy crops and is generally cultivated in arid climates and poor soils. Heat shock transcription factors (Hsfs) are key regulators of plant responses to abiotic and biotic stressors. However, their role and mechanism of action in switchgrass have not been elucidated. Hence, this study aimed to identify the Hsf family in switchgrass and understand its functional role in heat stress signal transduction and heat tolerance by using bioinformatics and RT-PCR analysis. Forty-eight PvHsfs were identified and divided into three main classes based on their gene structure and phylogenetic relationships: HsfA, HsfB, and HsfC. The results of the bioinformatics analysis showed a DNA-binding domain (DBD) at the N-terminal in PvHsfs, and they were not evenly distributed on all chromosomes except for chromosomes 8 N and 8 K. Many cis-elements related to plant development, stress responses, and plant hormones were identified in the promoter sequence of each PvHsf. Segmental duplication is the primary force underlying Hsf family expansion in switchgrass. The results of the expression pattern of PvHsfs in response to heat stress showed that PvHsf03 and PvHsf25 might play critical roles in the early and late stages of switchgrass response to heat stress, respectively, and HsfB mainly showed a negative response to heat stress. Ectopic expression of PvHsf03 in Arabidopsis significantly increased the heat resistance of seedlings. Overall, our research lays a notable foundation for studying the regulatory network in response to deleterious environments and for further excavating tolerance genes in switchgrass., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Wild panicgrass (Panicum miliaceum L. var. ruderale kit.) is an annual grass weed that primarily occurs in maize fields. Nicosulfuron is a widely used selective herbicide that effectively controls gramineous weeds in maize fields. However, owing to its long-term and extensive application, the control of P. miliaceum has been substantially reduced. The objective of this study was to determine the resistance pattern to ALS inhibitors in P. miliaceum and investigate the underlying resistance mechanisms. These are important for guiding the prevention and eradication of resistant weeds. Whole plant bioassays showed P. miliaceum had evolved high levels of resistance to nicosulfuron and multiple resistance to atrazine and mesotrione. The ALS gene sequence results indicated the absence of mutations in the resistant population. Additionally, there was no significant difference found in the inhibition rate of the ALS enzyme activity (I 50 ) between the resistant and sensitive populations. Following the application of malathion the resistant P. miliaceum population became more sensitive to nicosulfuron. At 96 h after application of nicosulfuron, glutathione-S-transferase activity in the resistant population was significantly higher than that in the susceptible population. The study reveals that the main cause of resistance to ALS inhibitor herbicide in P. miliaceum is likely increased metabolism of herbicides. These findings may assist in devising effective strategies for preventing and eliminating resistant P. miliaceum., Competing Interests: Declaration of Competing Interest No potential confilct of interest was reported by the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This work aimed to investigate the hypoglycemic effects and underlying mechanism of whole grain proso millet ( Panicum miliaceum L. ; WPM) on type 2 diabetes mellitus (T2DM). The results showed that WPM supplementation significantly reduced fasting blood glucose (FBG) and serum lipid levels in T2DM mice induced by a high-fat diet (HFD) combined with streptozotocin (STZ), with improved glucose tolerance, liver and kidney injury, and insulin resistance. In addition, WPM significantly inhibited the expression of gluconeogenesis-related genes G6pase , Pepck , Foxo1 , and Pgc -1α . Further study by miRNA high-throughput sequencing revealed that WPM supplementation mainly altered the liver miRNA expression profile of T2DM mice by increasing the expression of miR-144-3p&#95;R-1 and miR-423-5p, reducing the expression of miR-22-5p&#95;R-1 and miR-30a-3p. GO and KEGG analyses showed that the target genes of these miRNAs were mainly enriched in the PI3K/AKT signaling pathway. WPM supplementation significantly increased the level of PI3K, p-AKT, and GSK3β in the liver of T2DM mice. Taken together, WPM exerts antidiabetic effects by improving the miRNA profile and activating the PI3K/AKT signaling pathway to inhibit gluconeogenesis. This study implies that PM can act as a dietary supplement to attenuate T2DM.

Proso millet flour (PMF) and starch (PMS) were subjected to heat-moisture treatment (HMT) at 25 % moisture content and 110 °C for 4 h. The effects of HMT on physicochemical and structural properties and in vitro digestibility of PMF and PMS were analyzed. After HMT, SEM showed aggregation and damage to the surface of starch granules, while CLSM showed proteins wrapped around the granules. The amylopectin chain length distribution (CLD) remained unchanged in PMF and PMS after HMT, indicating intact covalent bonds between glucose units. HMT decreased the swelling power, solubility, viscosity of the paste, and gelatinization enthalpy and increased the pasting temperature and gelatinization temperature of PMF and PMS. HMT changed the XRD pattern of PMF from A to A + V type starches, whereas that of PMS remained unchanged. FTIR study showed an increase in the degree of short-range molecular order of PMF and PMS after HMT. In vitro digestibility evaluation showed that the rapidly (RDS) and slowly digestible starch (SDS) contents of PMF and PMS increased, whereas the resistant starch (RS) content decreased after HMT. HMT flour and starch have suitable properties for use in a wide range of food products, from canned to frozen, as well as non-food products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Key Message: PvLBD12 enhanced the salt tolerance by increasing proline accumulation, improving K + accumulation, and decreasing reactive oxygen species level in switchgrass. Abiotic stresses are the serious factors which limit plant development and productivity and restrict the agricultural economy. It is important, therefore, to understand the mechanism of abiotic tolerance in plants. Lateral organ boundaries domain (LBD) proteins as plant-specific transcription factors play important function in plant lateral organ development, plant regeneration, and abiotic stress. In our study, we identify 69 LBD members from switchgrass genome-wide sequences and classify them based on their homology with LBD proteins in Arabidopsis. RT-qPCR showed that PvLBD genes had different expression patterns under abiotic stress conditions, indicating that they play important roles in various stress. PvLBD12 was selected as a candidate gene for further functional analysis because it had the highest expression level under salt stress. Overexpression of PvLBD12 enhanced salt tolerance by altering a wide range of physiological responses (like increased proline accumulation, reduced malondialdehyde production, improved K + accumulation, and reduced Na + absorption) in switchgrass. Some stress response genes such as proline biosynthesis gene PvP5CS1, vacuolar Na + (K + )/H + antiporter gene PvNHX1, two key ROS-scavenging enzyme genes PvCAT and PvSOD were all upregulated in PvLBD12 overexpression lines. Taken together, PvLBD12 plays a pivotal role in response to salt stress by increasing proline accumulation, improving K + accumulation, reducing Na + absorption, and decreasing reactive oxygen species level. It will be better to understand the potential biological functions of LBD genes in other plants., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Switchgrass (Panicum virgatum L.), the prime bioenergy feedstock crop, is one ideal candidate for phytoremediation of cadmium (Cd). The absorption of Cd imposes severe endoplasmic reticulum (ER)-stress in plants. ER chaperone binding proteins (BiPs) are important modulators in ER-stress responses. The objective of this study was to characterize one Cd-responsive BiP gene, PvBiP2, in switchgrass for its roles in Cd tolerance and plant growth. PvBiP2 was up-regulated by Cd and the ER-stress inducer, dithiothreitol (DTT) and could be trans-activated by one Cd-responsive heat shock transcription factor PvHsfA4. Overexpression of PvBiP2 in switchgrass significantly increased its plant growth with higher height, stem diameter, leaf width, internode length, and tiller numbers than those of the wildtype (WT) plants under non-stress conditions. After 30 days of Cd treatment, the PvBiP2 over-expression transgenic lines showed 40-45% higher dry biomass accumulation with net photosynthesis rate (Pn), but lower electrolyte leakage (EL), malondialdehyde (MDA), and glutathione (GSH) levels than WT. Moreover, over-expressing PvBiP2 led to ∼90-140% Cd accumulation in plants but 46-57% lower Cd translocation rates to shoots. Together, the PvHSFA4-PvBiP2 module acted as positive regulators in plant Cd tolerance, and over-expressing PvBiP2 promoted plant vegetative growth as well as Cd tolerance making it an ideal molecular target for genetic improvement in switchgrass in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

In proso millet, in certain circumstances, drought stress greatly influences growth and metabolisms. Thus, the present study was aimed to examine morphological, biochemical and ROS mechanisms between plant and drought stress in Panicum miliaceum L. To create the drought condition, water irrigation was done at different time intervals including 4, 7, 10, 13 days and control. All the experiments were carried out at different maturity stages such as 30, 50, and 70 days (after sowing). The results demonstrated that the root length, proline, glycine betaine, amino acid and superoxide dismutase, catalase and peroxidase activities were boosted in all treatments as compared with control. As the proso millet matured, the length of shoots and the amount of chlorophyll pigment in the leaves reduced in all treatments as compared to control. Induced reduction of shoot growth, chlorophyll estimation and increases of root growth, osmolyte accumulations, antioxidant enzymes, were found to be drought-tolerant adaptative mechanisms in this study., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Understanding the effect of nitrogen fertilization on the quality of proso millet is key to expanding the use of this crop to address water scarcity and food security. Therefore, this study determined the impact of nitrogen fertilization on the proso millet quality. Nitrogen fertilization significantly increased the NR and GS activities and decreased the GBSSase activity, resulting in an increase in protein content and reduction in amylose content and L*, which decreased the appearance quality. Nitrogen fertilization increased the proportion of short amylopectin chains, resulting in a more disordered carbohydrate structure, and decreased the proportion of hydrophilic functional groups, contributing to an increase in setback viscosity and decrease in pasting temperature in the waxy (w139) variety. In contrast, the non-waxy (n297) variety exhibited a larger proportion of long amylopectin chains, lower ordered structure and hydrophobic functional groups after nitrogen fertilization, which strengthened the inter- and intramolecular forces of starch colloids., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

APX is a key antioxidant enzyme in higher plants, scavenging H 2 O 2 with ascorbate in several cellular compartments. Here, we report the crystal structures of cytosolic ascorbate peroxidase from switchgrass ( Panicum virgatum L., Pvi ), a strategic feedstock plant with several end uses. The overall structure of PviAPX was similar to the structures of other APX family members, with a bound ascorbate molecule at the ɣ-heme edge pocket as in other APXs. Our results indicated that the H 2 O 2 -dependent oxidation of ascorbate displayed positive cooperativity. Significantly, our study suggested that PviAPX can oxidize a broad range of phenylpropanoids with δ-meso site in a rather similar efficiency, which reflects its role in the fortification of cell walls in response to insect feeding. Based on detailed structural and kinetic analyses and molecular docking, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of PviAPX are proposed. Taken together, these observations shed new light on the function and catalysis of PviAPX, and potentially benefit efforts improve plant health and biomass quality in bioenergy and forage crops.

Cadmium (Cd) is a persistent heavy metal that poses environmental and public health concerns. This study aimed to identify the potential biomarkers responsible for Cd tolerance and accumulation by investigating the response of the content of essential metal elements, transporter gene expression, and root exudates to Cd stress in broomcorn millet (Panicum miliaceum). A hydroponics experiment was conducted using two broomcorn millet cultivars with distinct Cd tolerance levels and accumulation phenotypes (Cd-tolerant and Cd-sensitive cultivars). Cd stress inhibited lateral root growth, especially in the Cd-sensitive cultivar. Furthermore, Cd accumulation was significantly greater in the Cd-tolerant cultivar than in the Cd-sensitive cultivar. Cd stress significantly inhibited the absorption of essential metal elements and significantly increased the calcium concentration. Differentially expressed genes involved in metal ion transport were identified via transcriptome analysis. Cd stress altered the composition of root exudates, thus increasing lipid species and decreasing alkaloid, lignan, sugar, and alcohol species. Moreover, Cd stress significantly reduced most alkaloid, organic acid, and phenolic acid exudates in the Cd-tolerant cultivar, while it increased most lipid and phenolic acid exudates in the Cd-sensitive cultivar. Some significantly changed root exudates (ferulic acid, O-coumaric acid, and spermine) are involved in the phenylalanine biosynthesis, and arginine and proline metabolic pathways, thus, may be potential biomarkers of Cd stress response. Overall, metal ion absorption and root exudates are critical for Cd tolerance and accumulation in broomcorn millet. These findings provide valuable insights into improving Cd phytoremediation by applying mineral elements or metabolites., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The inclusion of warm-season grasses, such as switchgrass (Panicum virgatum) and eastern gamagrass (EG) (Tripsacum dactyloides), in vegetated buffer strips has been shown to mitigate herbicide contamination in runoff and increase herbicide degradation in soil. The mode of action by which buffer strip rhizospheres enhance herbicide degradation remains unclear, but microorganisms and phytochemicals are believed to facilitate degradation processes. The objectives of this study were to: 1) screen root extracts from seven switchgrass cultivars for the ability to degrade the herbicide atrazine (ATZ) in solution; 2) determine sorption coefficients (K d ) of the ATZ-degrading phytochemical 2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DBG) to soil and Ca-montmorillonite, and investigate if DBG or ATZ sorption alters degradation processes; and 3) quantify ATZ degradation rates and soil microbial response to ATZ application in mesocosms containing soil and select warm-season grasses. Phytochemicals extracted from the roots of switchgrass cultivars degraded 44-85% of ATZ in 16-h laboratory assays, demonstrating that some switchgrass cultivars could rapidly degrade ATZ under laboratory conditions. However, attempts to isolate ATZ-degrading phytochemicals from plant roots were unsuccessful. Sorption studies revealed that DBG was strongly sorbed to soil (K d  = 87.2 L kg -1 ) and Ca-montmorillonite (K d  = 31.7 L kg -1 ), and DBG driven hydrolysis of ATZ was entirely inhibited when either ATZ or DBG were sorbed to Ca-montmorillonite. Atrazine degradation rates in mesocosm soils were rapid (t 0.5  = 8.2-11.2 d), but not significantly different between soils collected from the two switchgrass cultivar mesocosms, the eastern gamagrass cultivar mesocosm, and the unvegetated mesocosm (control). Significant changes in three phospholipid fatty acid biomarkers were observed among the treatments. These changes indicated that different ATZ-degrading microbial consortia resulted in equivalent ATZ degradation rates between treatments. Results demonstrated that soil microbial response was the dominant mechanism controlling ATZ degradation in the soil studied, rather than root phytochemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Switchgrass (Panicum virgatum) is a bioenergy model crop valued for its energy efficiency and drought tolerance. The related monocot species rice (Oryza sativa) and maize (Zea mays) deploy species-specific, specialized metabolites as core stress defenses. By contrast, specialized chemical defenses in switchgrass are largely unknown. To investigate specialized metabolic drought responses in switchgrass, we integrated tissue-specific transcriptome and metabolite analyses of the genotypes Alamo and Cave-in-Rock that feature different drought tolerance. The more drought-susceptible Cave-in-Rock featured an earlier onset of transcriptomic changes and significantly more differentially expressed genes in response to drought compared to Alamo. Specialized pathways showed moderate differential expression compared to pronounced transcriptomic alterations in carbohydrate and amino acid metabolism. However, diterpenoid-biosynthetic genes showed drought-inducible expression in Alamo roots, contrasting largely unaltered triterpenoid and phenylpropanoid pathways. Metabolomic analyses identified common and genotype-specific flavonoids and terpenoids. Consistent with transcriptomic alterations, several root diterpenoids showed significant drought-induced accumulation, whereas triterpenoid abundance remained predominantly unchanged. Structural analysis verified select drought-responsive diterpenoids as oxygenated furanoditerpenoids. Drought-dependent transcriptome and metabolite profiles provide the foundation to understand the molecular mechanisms underlying switchgrass drought responses. Accumulation of specialized root diterpenoids and corresponding pathway transcripts supports a role in drought stress tolerance., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Fine tuning the progression of leaf senescence is important for plant fitness in nature, while the "staygreen" phenotype with delayed leaf senescence has been considered a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass (Panicum virgatum L.) CCCH-type Zinc finger gene, Strong Staygreen (PvSSG), was characterized as a suppressor of leaf senescence as overexpression or suppression of the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll (Chl) catabolic pathway genes were involved in the PvSSG-regulated leaf senescence. PvSSG was identified as a nucleus-localized protein with no transcriptional activity. By yeast two-hybrid screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1/2 (NAC-LIKE, ACTIVATED BY AP3/PI). Overexpression of PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating Chl catabolic genes to promote Chl degradation. PvSSG, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioning as a "brake" in the progression of leaf senescence. Moreover, overexpression of PvSSG resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass (Panicum virgatum), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass., Results: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession "Alamo", upland accession "Dacotah", and two upland/lowland F 1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors., Conclusion: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies., (© 2022. The Author(s).)

Background: Broomcorn millet is highly tolerant to drought and barren soil. Changes in chlorophyll content directly affect leaf color, which subsequently leadsleading to poor photosynthetic performance and reduced crop yield. Herein, we isolated a yellow leaf mutant (YX-yl) using a forward genetics approach and evaluated its agronomic traits, photosynthetic pigment content, chloroplast ultrastructure, and chlorophyll precursors. Furthermore, the molecular mechanism of yellowing was explored using transcriptome sequencing., Results: The YX-yl mutant showed significantly decreased plant height and low yield. The leaves exhibited a yellow-green phenotype and poor photosynthetic capacity during the entire growth period. The content of chlorophyll a, chlorophyll b, and carotenoids in YX-yl leaves was lower than that in wild-type leaves. Chlorophyll precursor analysis results showed that chlorophyll biosynthesis in YX-yl was hindered by the conversion of porphobilinogen to protoporphyrin IX. Examination of chloroplast ultrastructure in the leaves revealed that the chloroplasts of YX-yl accumulated on one side of the cell. Moreover, the chloroplast structure of YX-yl was degraded. The inner and outer membranes of the chloroplasts could not be distinguished well. The numbers of grana and grana thylakoids in the chloroplasts were low. The transcriptome of the yellowing mutant YX-yl was sequenced and compared with that of the wild type. Nine chlorophyll-related genes with significantly different expression profiles were identified: PmUROD, PmCPO, PmGSAM, PmPBDG, PmLHCP, PmCAO, PmVDE, PmGluTR, and PmPNPT. The proteins encoded by these genes were located in the chloroplast, chloroplast membrane, chloroplast thylakoid membrane, and chloroplast matrix and were mainly involved in chlorophyll biosynthesis and redox-related enzyme regulation., Conclusions: YX-yl is an ideal material for studying pigment metabolism mechanisms. Changes in the expression patterns of some genes between YX-yl and the wild type led to differences in chloroplast structures and enzyme activities in the chlorophyll biosynthesis pathway, ultimately resulting in a yellowing phenotype in the YX-yl mutant. Our findings provide an insight to the molecular mechanisms of leaf color formation and chloroplast development in broomcorn millet., (© 2022. The Author(s).)

Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late-acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild-type and miR156 overexpressing (miR156 OE ) switchgrass. Co-expression analysis, quantitative real-time polymerase chain reaction (qRT-PCR), transient dual luciferase assay, and chromatin immunoprecipitation-qPCR were all used to determine the activation of PvLBO by miR156-targeted Squamosa Promoter Binding Protein-like 2 (PvSPL2) in regulating tillering of switchgrass. PvLBOtranscripts dramatically declined in miR156 OE transgenic switchgrass, and the overexpression of PvLBO in the miR156 OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2. We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156-SPL module in switchgrass., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

S-adenosyl- l-methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S-adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plant laccase genes belong to a multigene family, play key roles in lignin polymerization, and participate in the resistance of plants to biotic and abiotic stresses. Switchgrass is an important resource for forage and bioenergy production, yet information about the switchgrass laccase gene family is scarce. Using bioinformatic approaches, a genome-wide analysis of the laccase multigene family in switchgrass was carried out in this study. In total, 49 laccase genes ( PvLac1 to PvLac49 ) were identified; these can be divided into five subclades, and 20 of them were identified as targets of miR397. The tandem and segmental duplication of laccase genes on Chr05 and Chr08 contributed to the expansion of the laccase family. The laccase proteins shared conserved signature sequences but displayed relatively low sequence similarity, indicating the potential functional diversity of switchgrass laccases. Switchgrass laccases exhibited distinct tissue/organ expression patterns, revealing that some laccases might be involved in the lignification process during stem development. All five of the laccase isoforms selected from different subclades responded to heavy metal. The immediate response of lignin-related laccases, as well as the delayed response of low-abundance laccases, to heavy-metal treatment shed light on the multiple roles of laccase isoforms in response to heavy-metal stress.

Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as important "monomers" for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Flowering means that the plant enters the reproductive growth stage, which is a crucial stage in the plant life cycle. Delaying flowering time to prolong vegetative growth is an important method to increase biomass yield and saccharification efficiency in switchgrass, It is of great significance to study the molecular mechanism of plant flowering and regulate the process of plant flowering in the process of biomass production. In this study, we identified 55 serine/threonine-protein kinase genes related to flower development from the switchgrass transcriptome database. Simultaneously, we cloned one of them, PvSTK1, whose expression level and differential fold were significantly higher than other members. PvSTK1 is located on chromosome 8N and its protein was in the cell membrane, cytoplasm, and nucleus. The spatio-temporal expression analysis of the PvSTK1 in switchgrass displayed that the PvSTK1 is crucial in vegetative period, however, not in the transition to reproductive period. Overexpression of PvSTK1 in Arabidopsis resulted in down-regulation of flower-promoting genes and up-regulation of flower-suppressing genes, thereby delaying flowering. In addition, PvSTK1 caused atrophy of the ovules of the florets at the base of the inflorescence, leading to sterility of the florets. The function of PvSTK1 is to inhibit the development of floral organs, and its overexpression can prolong its vegetative period. In the future, overexpression of the PvSTK1 gene in switchgrass will change the flowering time and increase yield and utilization efficiency of biomass., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

The perennial native switchgrass adapts better than other plant species do to marginal soils with low plant-available nutrients, including those with low phosphorus (P) content. Switchgrass roots and their associated microorganisms can alter the pools of available P throughout the whole soil profile making predictions of P availability in situ challenging. Plant P homeostasis makes monitoring of P limitation via measurements of plant P content alone difficult to interpret. To address these challenges, we developed a machine-learning model trained with high accuracy using the leaf tissue chemical profile, rather than P content. By applying this learned model in field trials across two sites with contrasting extractable soil P, we observed that actual plant available P in soil was more similar than expected, suggesting that adaptations occurred to alleviate the apparent P constraint. These adaptations come at a metabolic cost to the plant that have consequences for feedstock chemical components and quality. We observed that other biochemical signatures of P limitation, such as decreased cellulose-to-lignin ratios, were apparent, indicating re-allocation of carbon resources may have contributed to increased P acquisition. Plant P allocation strategies also differed across sites, and these differences were correlated with the subsequent year's biomass yields., (© 2022. The Author(s).)

Soil-borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole-genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor-like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor-root colonization by the ectomycorrhizal fungus Laccaria bicolor. By introducing PtLecRLK1 into a perennial grass known to be a non-host of L. bicolor, switchgrass (Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro-PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non-host to a host of L. bicolor. Furthermore, transcriptomic and proteomic analyses on inoculated-transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down-regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N-containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non-host to enhance plant productivity and fitness on marginal lands., (© 2021 Oak Ridge National laboratory. Plant Biotechnology Journal published by John Wiley & Sons Ltd.)

Little millet is a climate-resilient and high-nutrient value plant. The lack of molecular markers severely limits the adoption of modern genomic approaches in millet breeding studies. Here the transcriptome of three samples were sequenced. A total of 4443 genic-SSR motifs were identified in 30,220 unigene sequences. SSRs were found at a rate of 12.25 percent, with an average of one SSR locus per 10 kb. Among different repeat motifs, tri-nucleotide repeat (66.67) was the most abundant one, followed by di- (27.39P), and tetra- (3.83P) repeats. CDS contained fewer motifs with the majority of tri-nucleotides, while 3' and 5' UTR carry more motifs but have shorter repeats. Functional annotation of unigenes containing microsatellites, revealed that most of them were linked to metabolism, gene expression regulation, and response to environmental stresses. Fifty primers were randomly chosen and validated in five little millet and 20 minor millet genotypes; 48% showed polymorphism, with a high transferability (70%) rate. Identified microsatellites can be a noteworthy resource for future research into QTL-based breeding, genetic resource conservation, MAS selection, and evolutionary genetics., (© 2021. The Author(s).)

Broomcorn millet ( Panicum miliaceum L.) affected by smut (caused by the pathogen Sporisorium destruens ) has reduced production yields and quality. Determining the tolerance of broomcorn millet varieties is essential for smut control. This study focuses on the differences in the phenotypes, physiological characteristics, and transcriptomes of resistant and susceptible broomcorn millet varieties under Sporisorium destruens stress. In diseased broomcorn millet, the plant height and stem diameter were reduced, while the number of nodes increased. After infection, the activities of superoxide dismutase and peroxidase decreased, and malondialdehyde and relative chlorophyll content (SPAD) decreased. Transcriptome analysis showed 514 and 5452 differentially expressed genes (DEGs) in the resistant and susceptible varieties, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that pathways related to plant disease resistance, such as phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction, were significantly enriched. In addition, the transcriptome changes of cluster leaves and normal leaves in diseased broomcorn millet were analysed. Gene ontology and KEGG enrichment analyses indicated that photosynthesis played an important role in both varieties. These findings lay a foundation for future research on the molecular mechanism of the interaction between broomcorn millet and Sporisorium destruens .

Dinitrotoluene (DNT) has been extensively used in manufacturing munitions, polyurethane foams and other important chemical products. However, it is highly toxic and mutagenic to most organisms. Here, we synthesized a codon-optimized bacterial nitroreductase gene, NfsI, for plant expression. The kinetic analysis indicates that the recombinant NfsI can detoxify both 2,4-DNT and its sulfonate (DNTS), while it has a 97.6-fold higher catalytic efficiency for 2,4-DNT than DNTS. Furthermore, we overexpressed NfsI in switchgrass (Panicum virgatum L.), which is a multiple-purpose crop used for fodder and biofuel production as well as phytoremediation. The 2,4-DNT treatment inhibited root elongation of wild-type switchgrass plants and promoted reactive oxygen species (ROS) accumulation in roots. In contrast, overexpression of NfsI in switchgrass significantly alleviated 2,4-DNT-induced root growth inhibition and ROS overproduction. Thus, the NfsI overexpressing transgenic switchgrass plant removed 94.1% 2,4-DNT after 6 days, whose efficiency was 1.7-fold higher than control plants. Moreover, the comparative transcriptome analysis suggests that 22.9% of differentially expressed genes induced by 2,4-DNT may participate in NfsI-mediated 2,4-DNT detoxification in switchgrass. Our work sheds light on the function of NfsI during DNT phytoremediation for the first time, revealing the application potential of switchgrass plants engineered with NfsI., (© 2021 John Wiley & Sons Ltd.)

Improving plant biomass yield and/or feedstock quality for highly efficient lignocellulose conversion has been the main research focus in genetic modification of switchgrass (Panicum virgatum L.), a dedicated model plant for biofuel production. Here, we proved that overexpression of miR396 (OE-miR396) leads to reduced plant height and lignin content mainly by reducing G-lignin monomer content. We identified nineteen PvGRFs in switchgrass and proved thirteen of them were cleaved by miR396. MiR396-targeted PvGRF1, PvGRF9 and PvGRF3 showed significantly higher expression in stem. By separately overexpressing rPvGRF1, 3 and 9, in which synonymous mutations abolished the miR396 target sites, and suppression of PvGRF1/3/9 activity via PvGRF1/3/9-SRDX overexpression in switchgrass, we confirmed PvGRF1 and PvGRF9 played positive roles in improving plant height and G-lignin content. Overexpression of PvGRF9 was sufficient to complement the defective phenotype of OE-miR396 plants. MiR396-PvGRF9 modulates these traits partly by interfering GA and auxin biosynthesis and signalling transduction and cell wall lignin, glucose and xylan biosynthesis pathways. Moreover, by enzymatic hydrolysis analyses, we found that overexpression of rPvGRF9 significantly enhanced per plant sugar yield. Our results suggest that PvGRF9 can be utilized as a candidate molecular tool in modifying plant biomass yield and feedstock quality., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Lignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading., Results: The 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass (Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene (pShOMT) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process., Conclusion: This study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.

Sulfur (S) can play essential roles in protecting plants against abiotic stress, including heavy metal toxicity. However, the effect of this nutrient on plants exposed to barium (Ba) is still unknown. This study was designed to evaluate the S supply on oxidative stress and the antioxidant system of Tanzania guinea grass under exposure to Ba, grown in a nutrient solution under greenhouse conditions. It was studied the influence of S/Ba combinations in nutrient solution on oxidative stress indicators (hydrogen peroxide, malondialdehyde, and proline) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase). The treatments consisted in thirteen S/Ba combinations in the nutrient solution (0.1/0.0; 0.1/5.0; 0.1/20.0; 1.0/2.5; 1.0/10.0; 1.9/0.0 - control; 1.9/5.0; 1.9/20.0; 2.8/2.5; 2.8/10.0; 3.7/0.0; 3.7/5.0 and 3.7/20.0 mM of S and Ba, respectively). The plants were grown for two growth periods, which consisted of fourteen days of S supply and the eight days of Ba exposure each one. The severe S deficiency decreased the superoxide dismutase activity, regardless of Ba exposure in recently expanded leaves and culms plus sheaths. However, supplemental S supply (above 1.9 mM S, which corresponds to S supply adequate to plant growth) it improved the superoxide dismutase activity in these tissues under high Ba concentrations. Conversely, the severe S deficiency increased the activities of catalase, ascorbate peroxidase, and glutathione reductase in grass leaves slightly, without Ba exposure influence. It was observed that the supplemental S supply also induced the guaiacol peroxidase activity and proline production in culms plus sheaths under high Ba rates, showing values until 2.5 and 3.1 folds higher than the control treatment, respectively. In plants under exposure to 20.0 mM Ba, the supplemental S supply decreased the malondialdehyde content in culms plus sheaths in 17% compared to 1.9 mM S. These results indicate that supplemental S supply can mitigate Ba toxicity in Tanzania guinea grass, mainly by improving superoxide dismutase and guaiacol peroxidase activities, and proline metabolism., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Knowing how switchgrass (Panicum virgatum L.) responds and adapts to phosphorus (P)-limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P-stress on genome transcription and whole-plant metabolism, physiology and development in switchgrass. P-limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P-stress, and decreased root diameter with increased density and length of root hairs at severe P-stress. RNA-seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P-stress in roots and/or shoots compared to P-replete plants, with many stress-induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P-acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P-stress in shoots and roots, although this trend reversed under severe P-stress, especially in shoots., (© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

In this study, 95 accessions of proso millet (Panicum miliaceum L.) were characterized for starch physicochemical properties, including apparent amylose content (AAC), gel textural properties, Rapid Visco Analyzer (RVA) pasting viscosity properties, thermal and retrogradation properties. Based on genotypic data, the genetic diversity and inter-relationship of these starch traits were analyzed. Diverse starch quality was found, for example, AAC ranged from 0 to 32.3%, gelatinization temperature (GT) varied from 71.5 to 79.0 ℃, and RVA profile showed distinct patterns among proso millet of different AAC types. Interestingly, high AAC proso millet usually had GT lower than that of low AAC proso millet, which is different from the findings in rice starch. Many starch traits were significantly correlated and most of the 18 tested traits could be classified as either AAC-related traits or GT-related traits. In summary, the information presented here will be useful for further development of proso millet products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Proso millet is a highly nutritious cereal considered an essential component of processed foods. It is also recognized with high water-use efficiency as well as short growing seasons. This research was primarily aimed at investigating the genetic diversity among genotypes based on evaluating those important traits proposed in previous researches under both normal and salinity- stress conditions. Use of Amplified fragment length polymorphism (AFLP) molecular markers as well as evaluating the association between markers and the investigated traits under both conditions was also another purpose of this research., Results: According to the phenotypic correlation coefficients, the seed yield had the highest correlation with the forage and biological yields under both conditions. By disintegrating those traits investigated under normal and salinity-stress conditions into principal component analysis, it was found that the first four principal components justified more than 59.94 and 62.48% of the whole variance, respectively. The dendrogram obtained by cluster analysis displayed three groups of genotypes under both normal and salinity- stress conditions. Then, association analyses were conducted on 143 proso millet genotypes and 15 agronomic traits as well as 514 polymorphic AFLP markers (out of 866 created bands) generated by 11 primer combinations (out of the initial 20 primer combinations) EcoRI/MseI. The results obtained by mixed linear model (MLM) indicated that under normal conditions, the M14/E10-45 and M14/E10-60 markers had strong associations with seed yield. A similar trend was also observed for M14/E10-45 and M14/E11-44 markers in relation to forage yield. On the other hand, M14/E10-14, M14/E10-64 markers (for seed yield) and M14/E10-64 marker (for forage yield), had significant and stable association in all environments under salinity-stress conditions. Moreover, a number of markers showed considerable associations and stability under both normal and salinity stress conditions., Conclusions: According to the analysis of phenotypic data, the wide germplasm of Iranian proso millet has significant variation in terms of measured traits. It can be concluded that markers showing strong associations with traits under salinity-stress conditions are suitable candidates to be used in future marker-assisted selection (MAS) studies to improve salinity-resistance genotypes of Panicum miliaceum in arid and semiarid areas.

Switchgrass (Panicum virgatum L.) is an important crop for biofuel production but it also serves as host for greenbugs (Schizaphis graminum Rondani; GB). Although transcriptomic studies have been done to infer the molecular mechanisms of plant defense against GB, little is known about the effect of GB infestation on the switchgrass protein expression and phosphorylation regulation. The global response of the switchgrass cultivar Summer proteome and phosphoproteome was monitored by label-free proteomics shotgun in GB-infested and uninfested control plants at 10 days post infestation. Peptides matching a total of 3,594 proteins were identified and 429 were differentially expressed proteins in GB-infested plants relative to uninfested control plants. Among these, 291 and 138 were up and downregulated by GB infestation, respectively. Phosphoproteome analysis identified 310 differentially phosphorylated proteins (DP) from 350 phosphopeptides with a total of 399 phosphorylated sites. These phosphopeptides had more serine phosphorylated residues (79%), compared to threonine phosphorylated sites (21%). Overall, KEGG pathway analysis revealed that GB feeding led to the enriched accumulation of proteins important for biosynthesis of plant defense secondary metabolites and repressed the accumulation of proteins involved in photosynthesis. Interestingly, defense modulators such as terpene synthase, papain-like cysteine protease, serine carboxypeptidase, and lipoxygenase2 were upregulated at the proteome level, corroborating previously published transcriptomic data.

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (T opt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.