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.

Bioactive silver nanoparticles fabricated using Lasiurus scindicus and Panicum turgidum seed extracts: anticancer and antibacterial efficiency.

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 IC50 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.

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.

Transcriptomic analysis of ncRNAs and mRNAs interactions during drought stress in switchgrass.

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_miRNA480-mRNA, lncRNA-novel_miRNA304-mRNA, lncRNA/circRNA-novel_miRNA122-PvSS4, and lncRNA/circRNA-novel_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.

Enhanced metabolic ability enabled wild panicgrass (Panicum miliaceum L. var. ruderale kit.) resistance to ALS inhibitor herbicide.

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 (I50) 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.

Genome-wide identification, classification, and expression analysis of heat shock transcription factor family in switchgrass (Panicum virgatum L.).

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.

Whole Grain Proso Millet (Panicum miliaceum L.) Attenuates Hyperglycemia in Type 2 Diabetic Mice: Involvement of miRNA Profile.

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_R-1 and miR-423-5p, reducing the expression of miR-22-5p_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 GSK3ß 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.

Genome-wide characterization of LBD transcription factors in switchgrass (Panicum virgatum L.) and the involvement of PvLBD12 in salt tolerance.

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.

Effect of heat-moisture treatment on the physicochemical properties and digestibility of proso millet flour and starch.

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.

Cadmium tolerance and accumulation from the perspective of metal ion absorption and root exudates in broomcorn millet.

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.

Activity of Cytosolic Ascorbate Peroxidase (APX) from Panicum virgatum against Ascorbate and Phenylpropanoids.

APX is a key antioxidant enzyme in higher plants, scavenging H2O2 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 H2O2-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.

Nitrogen fertilizer affects starch synthesis to define non-waxy and waxy proso millet quality.

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.

Phenanthrene uptake and translocation by Panicum miliaceum L. tissues: an experimental study in an artificial environment.

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.

Influences of Natural Antioxidants, Reactive Oxygen Species and Compatible Solutes of Panicum Miliaceum L. Towards Drought Stress.

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.

Overexpression of PvBiP2 improved biomass yield and cadmium tolerance in switchgrass (Panicum virgatum L.).

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.

Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass).

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 F1 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.

Identification and function analysis of yellow-leaf mutant (YX-yl) of broomcorn millet.

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.

STRONG STAYGREEN inhibits DNA binding of PvNAP transcription factors during leaf senescence in switchgrass.

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.

Comparative transcriptomics and metabolomics reveal specialized metabolite drought stress responses in switchgrass (Panicum virgatum).

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.

Evaluating phytochemical and microbial contributions to atrazine degradation.

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 (Kd) of the ATZ-degrading phytochemical 2-ß-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 (Kd = 87.2 L kg-1) and Ca-montmorillonite (Kd = 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 (t0.5 = 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.