A pan-grass transcriptome reveals patterns of cellular divergence in crops.

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits1. Key traits that distinguish these species are mediated by specialized cell types2. Here we compare the transcriptomes of root cells in three grass species-Zea mays, Sorghum bicolor and Setaria viridis. We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others-driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.

Ground tissue circuitry regulates organ complexity in maize and Setaria.

Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of Arabidopsis. Here, we used single-cell RNA sequencing to rapidly generate a cell-resolution map of the maize root, revealing an alternative configuration of the tissue formative transcription factor SHORT-ROOT (SHR) adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue to elaborate anatomical complexity.

Wheat egg in vitro fusion with wheat and green bristlegrass sperm.

SummaryIsolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30-35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300-500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

Brassinosteroids Modulate Meristem Fate and Differentiation of Unique Inflorescence Morphology in Setaria viridis.

Inflorescence architecture is a key determinant of yield potential in many crops and is patterned by the organization and developmental fate of axillary meristems. In cereals, flowers and grain are borne from spikelets, which differentiate in the final iteration of axillary meristem branching. In Setaria spp, inflorescence branches terminate in either a spikelet or a sterile bristle, and these structures appear to be paired. In this work, we leverage Setaria viridis to investigate a role for the phytohormones brassinosteroids (BRs) in specifying bristle identity and maintaining spikelet meristem determinacy. We report the molecular identification and characterization of the Bristleless1 (Bsl1) locus in S. viridis, which encodes a rate-limiting enzyme in BR biosynthesis. Loss-of-function bsl1 mutants fail to initiate a bristle identity program, resulting in homeotic conversion of bristles to spikelets. In addition, spikelet meristem determinacy is altered in the mutants, which produce two florets per spikelet instead of one. Both of these phenotypes provide avenues for enhanced grain production in cereal crops. Our results indicate that the spatiotemporal restriction of BR biosynthesis at boundary domains influences meristem fate decisions during inflorescence development. The bsl1 mutants provide insight into the molecular basis underlying morphological variation in inflorescence architecture.

Spike-dip transformation of Setaria viridis.

Traditional method of Agrobacterium-mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium-mediated genetic transformation of S. viridis using spike dip. Pre-anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the ß-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5-day-old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025% Silwet L-77 and 200 µm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing ß-glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron-interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high-throughput transformation and potentially facilitates translational research in a monocot model plant.

Genetics and physiology of cell wall polysaccharides in the model C4 grass, Setaria viridis spp.

BACKGROUND: Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-ß-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. METHODS: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-ß-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-ß-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). RESULTS: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-ß-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-ß-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-ß-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-ß-glucan assays. CONCLUSION: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-ß-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.

Evolutionary convergence of cell-specific gene expression in independent lineages of C4 grasses.

Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r=0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons.

Phytolith analysis for differentiating between foxtail millet (Setaria italica) and green foxtail (Setaria viridis).

Foxtail millet (Setaria italica) is one of the oldest domesticated cereal crops in Eurasia, but identifying foxtail millets, especially in charred grains, and differentiating it from its wild ancestor, green foxtail (Setaria viridis), in the archaeobotanical remains, is still problematic. Phytolithic analysis provides a meaningful method for identifying this important crop. In this paper, the silicon structure patterns in the glumes, lemmas, and paleas from inflorescence bracts in 16 modern plants of foxtail millet and green foxtail from China and Europe are examined using light microscopy with phase-contrast and a microscopic interferometer. Our research shows that the silicon structure of ΩIII from upper lemmas and paleas in foxtail millet and green foxtail can be correspondingly divided into two groups. The size of ΩIII type phytolith of foxtail millet is bigger than that from green foxtail. Discriminant function analysis reveals that 78.4% of data on foxtail millet and 76.9% of data on green foxtail are correctly classified. This means certain morphotypes of phytoliths are relatively reliable tools for distinguishing foxtail millet from green foxtail. Our results also revealed that the husk phytolith morphologies of foxtail millets from China and Eastern Europe are markedly different from those from Western Europe. Our research gives a meaningful method of separating foxtail millet and green foxtail. The implications of these findings for understanding the history of foxtail millet domestication and cultivation in ancient civilizations are significant.