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

SUMMARY: Plasmodesmata (PD) are nanochannels that facilitate cell‐to‐cell transport in plants. More productive and photosynthetically efficient C4 plants form more PD at the mesophyll (M)‐bundle sheath (BS) interface in their leaves than their less efficient C3 relatives. In C4 leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO2 concentrating mechanism, which increases the CO2 partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C3 and C4 leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C3) and Setaria viridis (setaria, C4) with fluorescent protein‐tagged PD to build the first spatiotemporal atlas of leaf pit field (cluster of PD) density in monocots without the need for electron microscopy. Across leaf development, setaria had consistently more PD connections at the M‐BS wall interface than rice while the difference in M‐M pit field density varied. While light was a critical trigger of PD formation, cell type and function determined leaf pit field density. Complementary temporal mRNA sequencing and gene co‐expression network analysis revealed that the pattern of pit field density correlated with differentially expressed PD‐associated genes and photosynthesis‐related genes. PD‐associated genes identified from our co‐expression network analysis are related to cell wall expansion, translation and chloroplast signalling. Significance Statement: The important roles of plasmodesmata (PD) in plant biology present opportunity to improve crop performance but need more knowledge on the genetic control of PD formation to be realised. Here, we use unique bioimaging tools to understand PD formation during monocot leaf development in the context of photosynthesis, and identify potential genes involved. [ABSTRACT FROM AUTHOR]

Green foxtail is a troublesome weed in crop fields across China. A nova target HPPD inhibitor is widely used to control weeds in agricultural production. Mesotrione, an HPPD inhibitor, cannot control green foxtail effectively under the recommended field dose, indicating that green foxtail is tolerant to mesotrione. Interestingly, a green foxtail biotype that exhibits a greater tolerance to mesotrione (GR50 value 463.2 g ai ha−1) than that of the wild biotype (GR50 value 271.9 g ai ha−1) was found in Jilin Province, China. The HPPD genes isolated from the two biotypes genome were aligned, while no difference was found in the amino acid of the HPPD compared with that of the wild biotype. Through the qPCR experiment, the HPPD gene copy number variation and overexpression were also not found. Cytochrome P450 inhibitors (malathion and PBO), pretreatment, could effectively reverse the tolerance. Compared with the MT biotype, the in vivo activity of P450s was higher after the mesotrione treatment in the HT biotype. Therefore, P450s might be involved in the mechanism of tolerance. [ABSTRACT FROM AUTHOR]

Plant size is a critical component of agricultural productivity as larger plants produce more biomass. To identify genes related to plant size, we grouped C4 grasses into small and large and used OrthoFinder to find orthologous genes present in large but absent in small grasses. Three such genes were identified from sorghum (Sorghum bicolor [L.] Moench) by phylogenomic approach, and they encode nitrate transporter (Sobic.007G213200), oxysterol binding protein (SbRio.01G578800) and thioredoxin reductase (SbRio.05G168300), respectively. Overexpression of all three genes driven by the maize ubiquitin promoter in Setaria viridis (L.) Beauv. indicates that they all affected plant size as measured by plant height and tiller number. Both nitrate transporter and oxysterol binding protein increased plant height and tiller number, and thioredoxin reductase significantly decreased tiller number but had minimal effect on plant height. In rice (Oryza sativa L.), all three constructs reduced plant height significantly. The only commonality between the transgenic species was that nitrate transporter and oxysterol binding protein increased tiller number in both S. viridis and rice. Overall, we have demonstrated that phytogenomic approach can be used to identify genes responsible for large plant size in the grasses. [ABSTRACT FROM AUTHOR]

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

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

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

Maritime plants contain dwarf ecotypes that are adapted to exposure to strong winds transporting salt spray. When variants differentiated from local populations are well‐adapted and reproductively isolated, they become forerunners of ecotypes. This study aimed to determine how coastal variants arise from local populations under natural selection by studying phenotypic variations and survival of self‐fertilising Setaria viridis populations inhabiting pebble and sandy seashores in Japan.To identify adaptive traits characterising variants, common garden experiments were performed for determining the variation in morphology, salt spray tolerance and flowering time among populations in four adjacent habitats at two localities. To determine selective agents and their adaptation, field transplant experiments were performed, flowering phenology was monitored, and artificial crossing was conducted for F2 segregation.S. viridis populations comprised five coastal variants showing significantly higher salt spray tolerance than the inland variant. The most common of these was a short and tolerant variant adapted to cliff and pebble shore under intensive salt spray from the Pacific ocean. Three others were a tall and mid‐tolerant variant that endured mild breezes off the Seto inland sea, a tall and tolerant variant that survived sea splash from sporadic storms, and a prostrate and tolerant variant specialised for rock cracks exposed to strong sea winds. These variants imply that maritime plants first acquired salt spray tolerance for survival, after which compact plants evolved in habitats where strong winds caused damage from salt spray. Flowering in a fifth variant was delayed to avoid the fatal risk of reproducing during the mid‐summer drought. Although salt spray tolerance and late‐flowering were partially recessive, self‐fertilisation of S. viridis may have increased the homozygosity of recessive genes to generate novel variants.Synthesis. Diverse intensities of salt spray and wind as well as summer drought generated various coastal variants in parapatry and sympatry. This was partially attributable to self‐fertilisation. Among these, a compact variant adapted to common local seashores may become the forerunner of an ecotype. This study on the local adaptation in mosaic habitats will assist in determining adaptation modes of plants and the beginning of ecotype formation. [ABSTRACT FROM AUTHOR]

Green foxtail is a troublesome weed in crop fields across China. A nova target HPPD inhibitor is widely used to control weeds in agricultural production. Mesotrione, an HPPD inhibitor, cannot control green foxtail effectively under the recommended field dose, indicating that green foxtail is tolerant to mesotrione. Interestingly, a green foxtail biotype that exhibits a greater tolerance to mesotrione (GR50 value 463.2 g ai ha−1) than that of the wild biotype (GR50 value 271.9 g ai ha−1) was found in Jilin Province, China. The HPPD genes isolated from the two biotypes genome were aligned, while no difference was found in the amino acid of the HPPD compared with that of the wild biotype. Through the qPCR experiment, the HPPD gene copy number variation and overexpression were also not found. Cytochrome P450 inhibitors (malathion and PBO), pretreatment, could effectively reverse the tolerance. Compared with the MT biotype, the in vivo activity of P450s was higher after the mesotrione treatment in the HT biotype. Therefore, P450s might be involved in the mechanism of tolerance.

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

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

As in most plants, during their growth from immature to mature stages, the leaves of Setaria viridis, a model C4 bioenergy plant, have differential growth rates from the base (immature or growing) to the tip (most mature). In this study, we constructed a multi-segment C4 leaf metabolic model of S. viridis with two cell types (bundle sheath and mesophyll cells) across four leaf segments (base to tip). We incorporated differential growth rates for each leaf segment as constraints and integrated transcriptomic data as the objective function for our model simulation using flux balance analysis. The model was able to predict the exchanges of metabolites between immature and mature segments of the leaf and the distribution of the activities of biomass synthesis across those segments. Our model demonstrated the use of a modelling approach in studying the source–sink relationship within an organ and provided insights into the metabolic interactions across different parts of a leaf. [ABSTRACT FROM AUTHOR]

Invertebrate granivore communities can consume numerous weed seeds in cropland, but how this granivory influences weed recruitment over time in continuous no‐till systems is unknown. Weed and surface‐active granivore communities were determined in soybeans (Glycine max) over 3 years in eastern South Dakota. We examined seed removal rates of sentinel redroot pigweed, green foxtail, and lambsquarters (Chenopodium album) seeds and applied gut content analysis of granivores to determine which species consume immuno‐marked seeds of green foxtail (Setaria viridis). Weed stands with low diversity and high biomass production were associated with granivore community complexity. In turn, granivore community complexity was associated with weed communities with low diversity and high biomass in the subsequent growing season. Fall seed consumption, as measured by seed removal rates and gut content analysis of foxtail seed markers, was positively correlated with weed recruitment in the subsequent growing season. Seed specialists behaved differently toward focal weed species than the granivore community at large. Gryllus pennsylvanicus abundance was correlated with higher green foxtail consumption and removal rates. None of the other dominant green foxtail consumers revealed a similar set of responses. We propose a feedback mechanism whereby granivore community complexity in cropland leads to larger but fewer weeds over multiple years. Weed communities with these characteristics then lead to more complex granivore communities. If these relationships persist over time with minimal disturbance in continuous no‐till cropping systems without pesticides, this feedback loop could reduce weed cover. [ABSTRACT FROM AUTHOR]

Abstract Invertebrate granivore communities can consume numerous weed seeds in cropland, but how this granivory influences weed recruitment over time in continuous no‐till systems is unknown. Weed and surface‐active granivore communities were determined in soybeans (Glycine max) over 3 years in eastern South Dakota. We examined seed removal rates of sentinel redroot pigweed, green foxtail, and lambsquarters (Chenopodium album) seeds and applied gut content analysis of granivores to determine which species consume immuno‐marked seeds of green foxtail (Setaria viridis). Weed stands with low diversity and high biomass production were associated with granivore community complexity. In turn, granivore community complexity was associated with weed communities with low diversity and high biomass in the subsequent growing season. Fall seed consumption, as measured by seed removal rates and gut content analysis of foxtail seed markers, was positively correlated with weed recruitment in the subsequent growing season. Seed specialists behaved differently toward focal weed species than the granivore community at large. Gryllus pennsylvanicus abundance was correlated with higher green foxtail consumption and removal rates. None of the other dominant green foxtail consumers revealed a similar set of responses. We propose a feedback mechanism whereby granivore community complexity in cropland leads to larger but fewer weeds over multiple years. Weed communities with these characteristics then lead to more complex granivore communities. If these relationships persist over time with minimal disturbance in continuous no‐till cropping systems without pesticides, this feedback loop could reduce weed cover.

RNAi pathway is a derived pathway of RdDM pathway, proteins AGOs, DCLs and RDRs of which play important roles in plant growth and development and in responses to abiotic/biotic stresses. In order to study the sequence and structural characteristics of these three main proteins of the RNAi pathway in Setaria viridis, we identified 13 AGO genes, seven DCL genes and four RDR genes in S. viridis by comparative genomics. Further we predicted the protein subcellular localization, phylogenetic relationship and conserved domain of these proteins. Meanwhile, the transcriptome data were used to analyze the expression patterns of three types of genes families in different organs in 16 different growth stages and different growth conditions of S. viridis. Protein conserved domain analysis revealed that SvDCL3b and SvRDR3 were lack of important domains. Transcriptome analysis showed that SvAGO1b, SvDCL1a and SvRDR1 were highly expressed in each family, which might play a major role in the RNAi pathway, and the expression patterns of homologous genes of S. viridis and S. italica were basically the same.In conclusion, this study provides a preliminary theoretical basis for understanding the functions and roles of the three main genes of the RNAi pathway in regulating the epigenetic modification of S. viridis, and provides a reference for the molecular mechanism of domestication between S.viridis and S. italica. [ABSTRACT FROM AUTHOR]

Sulfate assimilation is an essential pathway of plant primary metabolism, regulated by the demand for reduced sulfur (S). The S-containing tripeptide glutathione (GSH) is the key signal for such regulation in Arabidopsis, but little is known about the conservation of these regulatory mechanisms beyond this model species. Using two model monocot species, C3 rice (Oryza sativa) and C4 Setaria viridis , and feeding of cysteine or GSH, we aimed to find out how conserved are the regulatory mechanisms described for Arabidopsis in these species. We showed that while in principle the regulation is similar, there are many species-specific differences. For example, thiols supplied by the roots are translocated to the shoots in rice but remain in the roots of Setaria. Cysteine and GSH concentrations are highly correlated in Setaria , but not in rice. In both rice and Setaria , GSH seems to be the signal for demand-driven regulation of sulfate assimilation. Unexpectedly, we observed cysteine oxidation to sulfate in both species, a reaction that does not occur in Arabidopsis. This reaction is dependent on sulfite oxidase, but the enzyme(s) releasing sulfite from cysteine still need to be identified. Altogether our data reveal a number of unique features in the regulation of S metabolism in the monocot species and indicate the need for using multiple taxonomically distinct models to better understand the control of nutrient homeostasis, which is important for generating low-input crop varieties. [ABSTRACT FROM AUTHOR]

Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. More productive and photosynthetically efficient C 4 plants form more PD at the mesophyll (M)-bundle sheath (BS) interface in their leaves than their less efficient C 3 relatives. In C 4 leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO 2 concentrating mechanism, which increases the CO 2 partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C 3 and C 4 leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C 3 ) and Setaria viridis (setaria, C 4 ) with fluorescent protein-tagged PD to build the first spatiotemporal atlas of leaf pit field (cluster of PD) density in monocots without the need for electron microscopy. Across leaf development, setaria had consistently more PD connections at the M-BS wall interface than rice while the difference in M-M pit field density varied. While light was a critical trigger of PD formation, cell type and function determined leaf pit field density. Complementary temporal mRNA sequencing and gene co-expression network analysis revealed that the pattern of pit field density correlated with differentially expressed PD-associated genes and photosynthesis-related genes. PD-associated genes identified from our co-expression network analysis are related to cell wall expansion, translation and chloroplast signalling., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Low temperature and end-of-day far-red (EOD-FR) light signaling are two key factors limiting plant production and geographical location worldwide. However, the transcriptional dynamics of EOD-FR light conditions during chilling stress remain poorly understood. Here, we performed a comparative RNA-Seq-based approach to identify differentially expressed genes (DEGs) related to EOD-FR and chilling stress in Setaria viridis. A total of 7911, 324, and 13431 DEGs that responded to low temperature, EOD-FR and these two stresses were detected, respectively. Further DEGs analysis revealed that EOD-FR may enhance cold tolerance in plants by regulating the expression of genes related to cold tolerance. The result of weighted gene coexpression network analysis (WGCNA) using 13431 nonredundant DEGs exhibited 15 different gene network modules. Interestingly, a CO-like transcription factor named BBX2 was highly expressed under EOD-FR or chilling conditions. Furthermore, we could detect more expression levels when EOD-FR and chilling stress co-existed. Our dataset provides a valuable resource for the regulatory network involved in EOD-FR signaling and chilling tolerance in C4 plants. [ABSTRACT FROM AUTHOR]

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

Water deficit is a major constraint for crops of economic importance in almost all agricultural regions. However, plants have an active defense system to adapt to these adverse conditions, acting in the reprogramming of gene expression responsible for encoding microRNAs (miRNAs). These miRNAs promote the regulation to the target gene expression by the post-transcriptional (PTGS) and transcriptional gene silencing (TGS), modulating several pathways including defense response to water deficit. The broader knowledge of the miRNA expression profile and its regulatory networks in response to water deficit can provide evidence for the development of new biotechnological tools for genetic improvement of several important crops. In this study, we used Setaria viridis accession A10.1 as a C4 model plant to widely investigate the miRNA expression profile in early responses to different levels of water deficit. Ecophysiological studies in Setaria viridis under water deficit and after rewatering demonstrated a drought tolerant accession, capable of a rapid recovery from the stress. Deep small RNA sequencing and degradome studies were performed in plants submitted to drought to identify differentially expressed miRNA genes and their predicted targets, using in silico analysis. Our findings showed that several miRNAs were differentially modulated in response to distinctive levels of water deficit and after rewatering. The predicted mRNA targets mainly corresponded to genes related to cell wall remodeling, antioxidant system and drought-related transcription factors, indicating that these genes are rapidly regulated in early responses to drought stress. The implications of these modulations are extensively discussed, and higher-effect miRNAs are suggested as major players for potential use in genetic engineering to improve drought tolerance in economically important crops, such as sugarcane, maize, and sorghum. [ABSTRACT FROM AUTHOR]

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

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C4 crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C4 crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms. [ABSTRACT FROM AUTHOR]

Key message: A combined transcriptomic and metabolic analysis of Setaria viridis leaves responding to aphid infestation was used to identify genes related to serotonin biosynthesis. Setaria viridis (green foxtail), a short life-cycle C4 plant in the Poaceae family, is the wild ancestor of Setaria italica (foxtail millet), a resilient crop that provides good yields in dry and marginal land. Although S. viridis has been studied extensively in the last decade, the molecular mechanisms of insect resistance in this species remain under-investigated. To address this issue, we performed a metabolic analysis of S. viridis and discovered that these plants accumulate the tryptophan-derived compounds tryptamine and serotonin. To elucidate the defensive functions of serotonin, Rhophalosiphum padi (bird cherry-oat aphids) were exposed to this compound, either by exogenous application to the plant medium or with artificial diet bioassays. In both cases, exposure to serotonin increased aphid mortality. To identify genes that are involved in serotonin biosynthesis, we conducted a transcriptome analysis and identified several predicted S. viridis tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes. Two candidate genes were ectopically expressed in Nicotiana tabacum, where SvTDC1 (Sevir.6G066200) had tryptophan decarboxylase activity, and SvT5H1 (Sevir.8G219600) had tryptamine hydroxylase activity. Moreover, the function of the SvTDC1 gene was validated using virus-induced gene silencing in S. italica, which caused a reduction in serotonin levels. This study provides the first evidence of serotonin biosynthesis in Setaria leaves. The biosynthesis of serotonin may play an important role in defense responses and could prove to be useful for developing more pest-tolerant Setaria italica cultivars. [ABSTRACT FROM AUTHOR]

Little is known about how community composition in the plant microbiome is affected by events in the life of a plant. For example, when the plant is exposed to soil, microbial communities may be an important factor in root community assembly. We conducted two experiments asking whether the composition of the root microbiota in mature plants could be determined by either the timing of root exposure to microbial communities or priority effects by early colonizing microbes. Timing of microbial exposure was manipulated through an inoculation experiment, where plants of different ages were exposed to a common soil inoculum. Priority effects were manipulated by challenging roots with established microbiota with an exogenous microbial community. Results show that even plants with existing microbial root communities were able to acquire new microbial associates, but that timing of soil exposure affected root microbiota composition for both bacterial and fungal communities in mature plants. Plants already colonized were only receptive to colonizers at 1 week post-germination. Our study shows that the timing of soil exposure in the early life stages of a plant is important for the development of the root microbiota in mature plants. [ABSTRACT FROM AUTHOR]

Little is known about how community composition in the plant microbiome is affected by events in the life of a plant. For example, when the plant is exposed to soil, microbial communities may be an important factor in root community assembly. We conducted two experiments asking whether the composition of the root microbiota in mature plants could be determined by either the timing of root exposure to microbial communities or priority effects by early colonizing microbes. Timing of microbial exposure was manipulated through an inoculation experiment, where plants of different ages were exposed to a common soil inoculum. Priority effects were manipulated by challenging roots with established microbiota with an exogenous microbial community. Results show that even plants with existing microbial root communities were able to acquire new microbial associates, but that timing of soil exposure affected root microbiota composition for both bacterial and fungal communities in mature plants. Plants already colonized were only receptive to colonizers at 1 week post-germination. Our study shows that the timing of soil exposure in the early life stages of a plant is important for the development of the root microbiota in mature plants.

Linkage mapping has been widely used to identify quantitative trait loci (QTL) in many plants and usually requires a time-consuming and labor-intensive fine mapping process to find the causal gene underlying the QTL. Previously, we described QTG-Finder, a machine-learning algorithm to rationally prioritize candidate causal genes in QTLs. While it showed good performance, QTG-Finder could only be used in Arabidopsis and rice because of the limited number of known causal genes in other species. Here we tested the feasibility of enabling QTG-Finder to work on species that have few or no known causal genes by using orthologs of known causal genes as the training set. The model trained with orthologs could recall about 64% of Arabidopsis and 83% of rice causal genes when the top 20% ranked genes were considered, which is similar to the performance of models trained with known causal genes. The average precision was 0.027 for Arabidopsis and 0.029 for rice. We further extended the algorithm to include polymorphisms in conserved non-coding sequences and gene presence/absence variation as additional features. Using this algorithm, QTG-Finder2, we trained and cross-validated Sorghum bicolor and Setaria viridis models. The S. bicolor model was validated by causal genes curated from the literature and could recall 70% of causal genes when the top 20% ranked genes were considered. In addition, we applied the S. viridis model and public transcriptome data to prioritize a plant height QTL and identified 13 candidate genes. QTL-Finder2 can accelerate the discovery of causal genes in any plant species and facilitate agricultural trait improvement.

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

Water deficits are responsible for countless agricultural losses. Among the affected crops, C4 plants are of special interest due to their high water and nitrogen use efficiency. Two accessions of Setaria viridis (Ast-1 and A10.1) with contrasting responses to water deficit were used in the current work to better understand the mechanisms behind drought tolerance in C4 species. Our results showed that although the A10.1 accession exhibited a reduced size and lower Rfd values in comparison to Ast-1, it had overall higher Fv/Fm and lower NPQ values in well-watered conditions. The water deficit induction was performed with PEG-8000 at the grain-filling stage using dehydration cycles. Analysis of physiological measurements showed the A10.1 accession as being more tolerant to multiple water deficit exposures. In addition, PCA identified a clear difference in the pattern of drought response of the accessions. Four drought marker genes previously described in the literature were chosen to evaluate the response at the molecular level: SvP5CS2 , SvDHN1 , SvNAC6 , and SvWRKY1. Besides confirming that Ast-1 is a more sensitive accession, the expression analysis revealed that SvNAC1 might better monitor drought stress, while SvWRKY1 was able to differentiate the two accessions. Distinct evolutionary histories of each accession may be behind their differences in response to water deficits. [Display omitted] - Setaria viridis accessions show distinct responses to cycles of water deficit. - The A10.1 accession shows higher tolerance to dehydration when compared to Ast-1. - Chlorophyll fluorescence measurements are able to monitor the effects of drought. - SvNAC1 is a good drought marker and SvWRKY1 differentiates the accessions. [ABSTRACT FROM AUTHOR]

A number of charred plant seeds were recovered from the Donghulin site by means of flotation. The site is located in suburban Beijing and dates from 11,000 to 9000 BP. A total of 14 charred grains of foxtail millet have been collected and identified as of the domesticated species (Setaria italica) according to morphological analysis. One grain of broomcorn millet was also identified. These are the earliest domesticated millet grains recovered by flotation, providing crucial archaeological evidence for understanding the timing, locations, and processes of millet domestication. Moreover, the charred seeds of Setaria viridis provide important clues for exploring the wild ancestral plants of foxtail millet and the domestication process. The results of flotation at the Donghulin site are important for understanding the origins of dryland agriculture in North China, which was predominated by millet farming. [ABSTRACT FROM AUTHOR]

Recently, the less Shattering1 gene (SvLes1), an MYB transcription factor on chromosome V, in Setaria viridis, was reported to control the degree of seed shattering within S. viridis. SvLes1-1 and SvLes1-2 are the wild type (high shattering) allele and the reduced shattering allele, respectively. In addition to these two alleles, the loss-of-function allele through a transposable-element (TE) insertion in exon 2 was found in foxtail millet, a domesticated type of S. viridis, and was designated as SiLes1-TE. This gene is considered to be a domestication gene in foxtail millet. We screened 131 accessions of foxtail millet and found that 16 landraces (12.2%) of foxtail millet do not have the TE, despite expression of the non-shattering phenotype. We sequenced the SvLes1 gene of these 16 accessions and classified them into three alleles, SvLes1-1, SvLes1-2, and a new allele, SvLes1-3. The geographical distribution of these three alleles was different, suggesting that foxtail millet domestication and differentiation are more complex than expected. [ABSTRACT FROM AUTHOR]

A fundamental limitation of photosynthetic carbon fixation is the availability of CO2. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of Setaria viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis.

Abstract Background C4 photosynthesis is a key domain of plant research with outcomes ranging from crop quality improvement, biofuel production and efficient use of water and nutrients. A metabolic network model of C4 “lab organism” Setaria viridis with extensive gene-reaction associations can accelerate target identification for desired metabolic manipulations and thereafter in vivo validation. Moreover, metabolic reconstructions have also been shown to be a significant tool to investigate fundamental metabolic traits. Results A mass and charge balance genome-scale metabolic model of Setaria viridis was constructed, which was tested to be able to produce all major biomass components in phototrophic and heterotrophic conditions. Our model predicted an important role of the utilization of NH 4+ $_{4}^{+}$ and NO 3− $_{3}^{-}$ ratio in balancing charges in plants. A multi-tissue extension of the model representing C4 photosynthesis was able to utilize NADP-ME subtype of C4 carbon fixation for the production of lignocellulosic biomass in stem, providing a tool for identifying gene associations for cellulose, hemi-cellulose and lignin biosynthesis that could be potential target for improved lignocellulosic biomass production. Besides metabolic engineering, our modeling results uncovered a previously unrecognized role of the 3-PGA/triosephosphate shuttle in proton balancing. Conclusions A mass and charge balance model of Setaria viridis, a model C4 plant, provides the possibility of system-level investigation to identify metabolic characteristics based on stoichiometric constraints. This study demonstrated the use of metabolic modeling in identifying genes associated with the synthesis of particular biomass components, and elucidating new role of previously known metabolic processes.

SUMMARY: C4 photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO2 concentration at the site of CO2 fixation. C4 plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP‐dependent malic enzyme subtype, focussing on cell‐specific electron transport capacity. Plants grown under low light (LL) maintained CO2 assimilation rates similar to high light plants but had an increased chlorophyll and light‐harvesting‐protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen‐evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b6f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII – light‐harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C4 plants and will support the development of strategies for crop improvement, including the engineering of C4 photosynthesis into C3 plants. Significance Statement: The efficiency of C4 photosynthesis decreases under low irradiance, causing a loss of productivity in crop canopies. We investigate shade acclimation of a model monocot of NADP‐dependent malic enzyme subtype, analysing cell‐specific protein expression and electron transport capacity. We demonstrate that C4 plants adjust the abundance and activity of Photosystem II in response to irradiance, specifically in bundle sheath cells. [ABSTRACT FROM AUTHOR]

PIRINs (PRNs) comprise a family of cupin domain-containing proteins that are conserved between prokaryotic and eukaryotic organisms, but whose functions remain largely elusive. A member of the PIRIN family has been recently characterized as a transcriptional co-regulator of lignin deposition in the eudicot Arabidopsis thaliana, consisting of another player in the complex regulatory mechanism controlling the timing, localization and final structure of the lignin polymer. However, a similar regulatory function for PRNs in other plant species remains to be determined. Here, we characterized the PIRIN gene family in the C4 model grass Setaria viridis and further identified members potentially involved in lignification. A total of 4 genes encoding PRNs (SvPRN1 to SvPRN4) were found in the genome of S. viridis, similar to the number found in other grasses. Phylogenetic analysis using 39 plant species depicted three groups of PRN proteins, not equally conserved across the plant kingdom. SvPRN2 clustered into Group 1 together with other PRNs from eudicots and grasses, whereas the other SvPRNs clustered into Group 3, consisting mainly of PRNs from grasses. Group 2 was exclusively composed of PRN proteins from eudicot species, together with PRN from the basal Angiosperm Amborella trichopoda. Predictive structural analysis suggests that PRN proteins belonging to Group 3 harbor distinct structural features potentially related to functional diversification. The identification of SvPRN genes potentially involved in lignification in S. viridis was based on the following criteria: i) similar expression pattern to lignin biosynthetic genes in a set of different S. viridis tissues; ii) similar expression pattern to lignin biosynthetic genes in the elongating internode of S. viridis; iii) co-expression with secondary cell wall- (SCW) and lignin-related genes in co-expression databases; iv) presence of SCW-related cis elements in its promoter region; v) high expression in S. viridis tissues undergoing active lignification. These analyses suggest that SvPRN2 is the strongest candidate to play a role in the regulation of lignin deposition in S. viridis, although other members of the family met some of these criteria and might also function in this biological process. Functional studies are necessary to confirm and further characterize the precise role of these SvPRN genes in S. viridis. [ABSTRACT FROM AUTHOR]

Globally, most human caloric intake is from crops that belong to the grass family (Poaceae), including sugarcane (Saccharum spp.), rice (Oryza sativa), maize (or corn, Zea mays), and wheat (Triticum aestivum). The grasses have a unique morphology and inflorescence architecture, and some have also evolved an uncommon photosynthesis pathway that confers drought and heat tolerance, the C4 pathway. Most secondary-level students are unaware of the global value of these crops and are unfamiliar with plant science fundamentals such as grass architecture and the genetic concepts of genotype and phenotype. Green foxtail millet (Setaria viridis) is a model organism for C4 plants and a close relative of globally important grasses, including sugarcane. It is ideal for teaching about grass morphology, the economic value of grasses, and the C4 photosynthetic pathway. This article details a teaching module that uses S. viridis to engage entire classrooms of students in authentic research through a laboratory investigation of grass morphology, growth cycle, and genetics. This module includes protocols and assignments to guide students through the process of growing one generation of S. viridis mutants and reference wild-type plants from seed to seed, taking measurements, making critical observations of mutant phenotypes, and discussing their physiological implications. [ABSTRACT FROM AUTHOR]

In C4 species, β-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3–, the substrate used by phospho enol pyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO 2 -responsive mutant 1 (lcr1) isolated from an N -nitroso- N -methylurea- (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.5G247800 of lcr1 possessed a single nucleotide transition from cytosine to thymine in a β-CA gene causing an amino acid change from leucine to phenylalanine. This resulted in severe reduction in growth and photosynthesis in the mutant. Both the CO2 compensation point and carbon isotope discrimination values of the mutant were significantly increased. Growth of the mutants was stunted when grown under ambient p CO2 but recovered at elevated p CO2. Further bioinformatics analyses revealed that the mutation has led to functional changes in one of the conserved residues of the protein, situated near the catalytic site. CA transcript accumulation in the mutant was 80% lower, CA protein accumulation 30% lower, and CA activity ~98% lower compared with the wild type. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEPC protein was significantly increased and accumulation of malate dehydrogenase and malic enzyme decreased. The reduction of CA protein activity and abundance in lcr1 restricts the supply of bicarbonate to PEPC, limiting C4 photosynthesis and growth. This study establishes Sevir.5G247800 as the major CA allele in Setaria for C4 photosynthesis and provides important insights into the function of CA in C4 photosynthesis that would be required to generate a rice plant with a functional C4 biochemical pathway. [ABSTRACT FROM AUTHOR]

SUMMARY: The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA‐binding domain found in a plant‐specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole‐plant chlorotic phenotypes. Co‐immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes. Significance Statement: The CRM proteins are a plant‐specific family of RNA‐binding proteins that are involved in intron splicing in chloroplasts and mitochondria. We describe the role of a previously uncharacterized member, CFM1, in chloroplast group II intron splicing in Setaria viridis. [ABSTRACT FROM AUTHOR]

Heterologous overexpression of Arabidopsis cellulase 1 (Atcel1) results in enhanced yield, early maturity, and increased biomass in dicotyledonous species like poplar and eucalyptus but has not been demonstrated in monocots. We produced transgenic Setaria viridis accession A10.1 plants overexpressing a monocotyledonous codon optimized (MCO) Atcel1. Agronomic characterization of the transgenic events showed that heterologous overexpression of MCOAtcel1 caused enhanced grain yield, shoot biomass, and accelerated maturation rate in the model grass species S. viridis under growth chamber conditions. The agronomic trait differences observed were consistent with previous reports in dicots but are here described in a monocot species and associated with increased seed yield. Overexpression of Atcel1 in S. viridis was shown to increase the number of panicles and seeds by 24–30%, enhance overall grain yield by up to 26%, and lead to a shoot dry biomass increase of 16–19%. Overexpression also reduced time to plant maturation and senescence by 12.5%. Our findings in S. viridis suggest that manipulation of Atcel1 has potential for developing early-maturing and higher-yielding monocotyledonous biomass crops suitable for climate-smart agriculture.

The present study reveals contrasting responses of photosynthesis to salt stress in two C4 species: a glycophyte Setaria viridis (SV) and a halophyte Spartina alterniflora (SA). Specifically, the effect of short-term salt stress treatment on the photosynthetic CO2 uptake and electron transport were investigated in SV and its salt-tolerant close relative SA. In this experiment, at the beginning, plants were grown in soil then were exposed to salt stress under hydroponic conditions for two weeks. SV demonstrated a much higher susceptibility to salt stress than SA; while, SV was incapable to survive subjected to about 100 mM, SA can tolerate salt concentrations up to 550 mM with slight effect on photosynthetic CO2 uptake rates and electrons transport chain conductance (gETC). Regardless the oxygen concentration used, our results show an enhancement in the P700 oxidation with increasing O2 concentration for SV following NaCl treatment and almost no change for SA. We also observed an activation of the cyclic NDH-dependent pathway in SV by about 2.36 times upon exposure to 50 mM NaCl for 12 days (d); however, its activity in SA drops by about 25% compared to the control without salt treatment. Using PTOX inhibitor (n-PG) and that of the Qo-binding site of Cytb6/f (DBMIB), at two O2 levels (2 and 21%), to restrict electrons flow towards PSI, we successfully revealed the presence of a possible PTOX activity under salt stress for SA but not for SV. However, by q-PCR and western-blot analysis, we showed an increase in PTOX amount by about 3–4 times for SA under salt stress but not or very less for SV. Overall, this study provides strong proof for the existence of PTOX as an alternative electron pathway in C4 species (SA), which might play more than a photoprotective role under salt stress.

Setaria viridis (green foxtail) has been identified as a potential experimental model system to genetically and molecularly characterise the C4 monocotyledonous grasses due to its small physical size, short generation time and prolific seed production, together with a sequenced and annotated genome. Setaria viridis is the wild ancestor of the cropping species, foxtail millet (Setaria italica), with both Setaria species sharing a close evolutionary relationship with the agronomically important species, maize, sorghum, and sugarcane, as well as the bioenergy feedstocks, switchgrass, and Miscanthus. However, an efficient and reproducible transformation protocol is required to further advance the use of S. viridis to study the molecular genetics of C4 monocotyledonous grasses. An efficient and reproducible protocol was established for Agrobacterium tumefaciens-mediated transformation of S. viridis (Accession A10) regenerable callus material derived from mature seeds, a protocol that returned an average transformation efficiency of 6.3%. The efficiency of this protocol was the result of the: (i) use of mature embryo derived callus material; (ii) age of the seed used to induce callus formation; (iii) composition of the callus induction media, including the addition of the ethylene inhibitor, silver nitrate; (iv) use of a co-cultivation approach, and; (v) concentration of the selective agent. Our protocol furthers the use of S. viridis as an experimental model system to study the molecular genetics of C4 monocotyledonous grasses for the potential future development of improved C4 cropping species.

Abstract Background Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Results Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues. Conclusions This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species.

SUMMARY: Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate‐induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale‐green seedlings in the M3 generation, and a subset of these was identified to be deficient in post‐transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing‐based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA‐sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid‐specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast‐encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale‐green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3′ end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model. Significance Statement: Deep understanding of photosynthetic mechanisms in C4 plants is relevant to evolutionary, biochemical and applied aspects of plant science. Setaria viridis is an emerging C4 model system, and we present the first study of mutants defective in chloroplast functions required for photosynthesis. [ABSTRACT FROM AUTHOR]