Genetic control of branching patterns in grass inflorescences.

Inflorescence branching in the grasses controls the number of florets and hence the number of seeds. Recent data on the underlying genetics come primarily from rice and maize, although new data are accumulating in other systems as well. This review focuses on a window in developmental time from the production of primary branches by the inflorescence meristem through to the production of glumes, which indicate the transition to producing a spikelet. Several major developmental regulatory modules appear to be conserved among most or all grasses. Placement and development of primary branches are controlled by conserved auxin regulatory genes. Subtending bracts are repressed by a network including TASSELSHEATH4, and axillary branch meristems are regulated largely by signaling centers that are adjacent to but not within the meristems themselves. Gradients of SQUAMOSA-PROMOTER BINDING-like and APETALA2-like proteins and their microRNA regulators extend along the inflorescence axis and the branches, governing the transition from production of branches to production of spikelets. The relative speed of this transition determines the extent of secondary and higher order branching. This inflorescence regulatory network is modified within individual species, particularly as regards formation of secondary branches. Differences between species are caused both by modifications of gene expression and regulators and by presence or absence of critical genes. The unified networks described here may provide tools for investigating orphan crops and grasses other than the well-studied maize and rice.

Conserved noncoding sequences provide insights into regulatory sequence and loss of gene expression in maize.

Thousands of species will be sequenced in the next few years; however, understanding how their genomes work, without an unlimited budget, requires both molecular and novel evolutionary approaches. We developed a sensitive sequence alignment pipeline to identify conserved noncoding sequences (CNSs) in the Andropogoneae tribe (multiple crop species descended from a common ancestor ∼18 million years ago). The Andropogoneae share similar physiology while being tremendously genomically diverse, harboring a broad range of ploidy levels, structural variation, and transposons. These contribute to the potential of Andropogoneae as a powerful system for studying CNSs and are factors we leverage to understand the function of maize CNSs. We found that 86% of CNSs were comprised of annotated features, including introns, UTRs, putative cis-regulatory elements, chromatin loop anchors, noncoding RNA (ncRNA) genes, and several transposable element superfamilies. CNSs were enriched in active regions of DNA replication in the early S phase of the mitotic cell cycle and showed different DNA methylation ratios compared to the genome-wide background. More than half of putative cis-regulatory sequences (identified via other methods) overlapped with CNSs detected in this study. Variants in CNSs were associated with gene expression levels, and CNS absence contributed to loss of gene expression. Furthermore, the evolution of CNSs was associated with the functional diversification of duplicated genes in the context of maize subgenomes. Our results provide a quantitative understanding of the molecular processes governing the evolution of CNSs in maize.

Grain shattering by cell death and fracture in Eragrostis tef.

Abscission, known as shattering in crop species, is a highly regulated process by which plants shed parts. Although shattering has been studied extensively in cereals and a number of regulatory genes have been identified, much diversity in the process remains to be discovered. Teff (Eragrostis tef) is a crop native to Ethiopia that is potentially highly valuable worldwide for its nutritious grain and drought tolerance. Previous work has suggested that grain shattering in Eragrostis might have little in common with other cereals. In this study, we characterize the anatomy, cellular structure, and gene regulatory control of the abscission zone (AZ) in E. tef. We show that the AZ of E. tef is a narrow stalk below the caryopsis, which is common in Eragrostis species. X-ray microscopy, scanning electron microscopy, transmission electron microscopy, and immunolocalization of cell wall components showed that the AZ cells are thin walled and break open along with programmed cell death (PCD) at seed maturity, rather than separating between cells as in other studied species. Knockout of YABBY2/SHATTERING1, documented to control abscission in several cereals, had no effect on abscission or AZ structure in E. tef. RNA sequencing analysis showed that genes related to PCD and cell wall modification are enriched in the AZ at the early seed maturity stage. These data show that E. tef drops its seeds using a unique mechanism. Our results provide the groundwork for understanding grain shattering in Eragrostis and further improvement of shattering in E. tef.

Brachypodium distachyon as a Genetic Model System.

Brachypodium distachyon has emerged as a powerful model system for studying the genetics of flowering plants. Originally chosen for its phylogenetic proximity to the large-genome cereal crops wheat and barley, it is proving to be useful for more than simply providing markers for comparative mapping. Studies in B. distachyon have provided new insight into the structure and physiology of plant cell walls, the development and chemical composition of endosperm, and the genetic basis for cold tolerance. Recent work on auxin transport has uncovered mechanisms that apply to all angiosperms other than Arabidopsis. In addition to the areas in which it is currently used, B. distachyon is uniquely suited for studies of floral development, vein patterning, the controls of the perennial versus annual habit, and genome organization.

The YABBY gene SHATTERING1 controls activation rather than patterning of the abscission zone in Setaria viridis.

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.

Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

Sterile Spikelets Contribute to Yield in Sorghum and Related Grasses.

Sorghum (Sorghum bicolor) and its relatives in the grass tribe Andropogoneae bear their flowers in pairs of spikelets in which one spikelet (seed-bearing or sessile spikelet [SS]) of the pair produces a seed and the other is sterile or male (staminate). This division of function does not occur in other major cereals such as wheat (Triticum aestivum) or rice (Oryza sativa). Additionally, one bract of the SS spikelet often produces a long extension, the awn, that is in the same position as, but independently derived from, that of wheat and rice. The function of the sterile spikelet is unknown and that of the awn has not been tested in Andropogoneae. We used radioactive and stable isotopes of carbon, RNA sequencing of metabolically important enzymes, and immunolocalization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to show that the sterile spikelet assimilates carbon, which is translocated to the largely heterotrophic SS. The awn shows no evidence of photosynthesis. These results apply to distantly related species of Andropogoneae. Removal of sterile spikelets in sorghum significantly decreases seed weight (yield) by ∼9%. Thus, the sterile spikelet, but not the awn, affects yield in the cultivated species and fitness in the wild species.

Diverse ecological functions and the convergent evolution of grass awns.

The awn of grasses is a long, conspicuous outgrowth of the floral bracts in a grass spikelet. It is known to impact agricultural yield, but we know little about its broader ecological function, nor the selective forces that lead to its evolution. Grass awns are phenotypically diverse across the extant ~12,000 species of Poaceae. Awns have been lost and gained repeatedly over evolutionary time, between and within lineages, suggesting that they could be under selection and might provide adaptive benefit in some environments. Despite the phylogenetic context, we know of no studies that have tested whether the origin of awns correlates with putative selective forces on their form and function. Presence or absence of awns is not plastic; rather, heritability is high. The awns of grasses often are suggested as adaptations for dispersal, and most experimental work has been aimed at testing this hypothesis. Proposed dispersal functions include soil burial, epizoochory, and aerial orientation. Awns may also protect the seed from drought, herbivores, or fire by helping it become buried in soil. We do not fully understand the fitness or nutrient costs of awn production, but in some species awns function in photosynthesis, providing carbon to the seed. Here we show that awns likely provide an adaptive advantage, but argue that studies on awn function have lacked critical phylogenetic information to demonstrate adaptive convergent evolution, are taxonomically biased, and often lack clear alternative hypotheses.

Hybridization, polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum).

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P. vaginatum, we employed genotyping-by-sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing > 170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P. vaginatum differ in their adaptive strategies. The fine-textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse-textured ecotype consists largely of clonally-propagating triploid and diploid genotypes. The coarse-textured ecotype appears to be derived from hybridization between fine-textured P. vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine-textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P. vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.

Continued Adaptation of C4 Photosynthesis After an Initial Burst of Changes in the Andropogoneae Grasses.

C$_{4}$ photosynthesis is a complex trait that sustains fast growth and high productivity in tropical and subtropical conditions and evolved repeatedly in flowering plants. One of the major C$_{4}$ lineages is Andropogoneae, a group of $\sim $1200 grass species that includes some of the world's most important crops and species dominating tropical and some temperate grasslands. Previous efforts to understand C$_{4}$ evolution in the group have compared a few model C$_{4}$ plants to distantly related C$_{3}$ species so that changes directly responsible for the transition to C$_{4}$ could not be distinguished from those that preceded or followed it. In this study, we analyze the genomes of 66 grass species, capturing the earliest diversification within Andropogoneae as well as their C$_{3}$ relatives. Phylogenomics combined with molecular dating and analyses of protein evolution show that many changes linked to the evolution of C$_{4}$ photosynthesis in Andropogoneae happened in the Early Miocene, between 21 and 18 Ma, after the split from its C$_{3}$ sister lineage, and before the diversification of the group. This initial burst of changes was followed by an extended period of modifications to leaf anatomy and biochemistry during the diversification of Andropogoneae, so that a single C$_{4}$ origin gave birth to a diversity of C$_{4}$ phenotypes during 18 million years of speciation events and migration across geographic and ecological spaces. Our comprehensive approach and broad sampling of the diversity in the group reveals that one key transition can lead to a plethora of phenotypes following sustained adaptation of the ancestral state. [Adaptive evolution; complex traits; herbarium genomics; Jansenelleae; leaf anatomy; Poaceae; phylogenomics.].

Divergent gene expression networks underlie morphological diversity of abscission zones in grasses.

Abscission is a process in which plants shed their parts, and is mediated by a particular set of cells, the abscission zone (AZ). In grasses (Poaceae), the position of the AZ differs among species, raising the question of whether its anatomical structure and genetic control are conserved. The ancestral position of the AZ was reconstructed. A combination of light microscopy, transmission electron microscopy, RNA-Seq analyses and RNA in situ hybridisation were used to compare three species, two (weedy rice and Brachypodium distachyon) with the AZ in the ancestral position and one (Setaria viridis) with the AZ in a derived position below a cluster of flowers (spikelet). Rice and Brachypodium are more similar anatomically than Setaria. However, the cell wall properties and the transcriptome of rice and Brachypodium are no more similar to each other than either is to Setaria. The set of genes expressed in the studied tissues is generally conserved across species, but the precise developmental and positional patterns of expression and gene networks are almost entirely different. Transcriptional regulation of AZ development appears to be extensively rewired among the three species, leading to distinct anatomical and morphological outcomes.

Comprehensive 3D phenotyping reveals continuous morphological variation across genetically diverse sorghum inflorescences.

●Inflorescence architecture in plants is often complex and challenging to quantify, particularly for inflorescences of cereal grasses. Methods for capturing inflorescence architecture and for analyzing the resulting data are limited to a few easily captured parameters that may miss the rich underlying diversity. ●Here, we apply X-ray computed tomography combined with detailed morphometrics, offering new imaging and computational tools to analyze three-dimensional inflorescence architecture. To show the power of this approach, we focus on the panicles of Sorghum bicolor, which vary extensively in numbers, lengths, and angles of primary branches, as well as the three-dimensional shape, size, and distribution of the seed. ●We imaged and comprehensively evaluated the panicle morphology of 55 sorghum accessions that represent the five botanical races in the most common classification system of the species, defined by genetic data. We used our data to determine the reliability of the morphological characters for assigning specimens to race and found that seed features were particularly informative. ●However, the extensive overlap between botanical races in multivariate trait space indicates that the phenotypic range of each group extends well beyond its overall genetic background, indicating unexpectedly weak correlation between morphology, genetic identity, and domestication history.

The anatomy of abscission zones is diverse among grass species.

PREMISE: Abscission zones (AZ) are specialized cell layers that separate plant parts at the organ junction upon developmental or environmental signals. Fruit or seed abscission has been well studied in model species because of its crucial role for seed dispersal. Previous work showed that AZ localization differs among species of Poaceae and that AZ formation is histologically and genetically distinct in three distantly related grass species, refuting the idea of a broadly conserved module. However, whether AZ structure is consistent within subfamilies is unknown. METHODS: Eleven species were selected from six subfamilies of Poaceae, and their AZ was investigated using paraffin-embedded, stained material. Observations were added from the literature for an additional six species. Data were recorded on AZ location and whether cells in the AZ were distinguishable by size or lignification. Characteristics of the AZ were mapped on the phylogeny using maximum likelihood. RESULTS: Abscission zone anatomy and histology vary among species, and characteristics of the AZ do not correlate with phylogeny. Twelve of the seventeen studied species have an AZ in which the cells are significantly smaller than surrounding cells. Of these, eight have differential lignification. Differential lignification is often associated with differential cell size, but not vice versa. CONCLUSIONS: Neither smaller cells in the AZ nor differential lignification between the AZ and surrounding cells is required for abscission, although differential cell size and lignification are often correlated. Abscission zone anatomy does not correlate with phylogeny, suggesting its rapid change over evolutionary time.

Getting closer: vein density in C4 leaves.

Contents Summary 1260 I. Introduction 1260 II. Molecular and genetic mechanisms of C4 leaf venation 1262 III. Conclusions and future perspectives 1266 Acknowledgements 1266 References 1266 SUMMARY: C4 grasses are major contributors to the world's food supply. Their highly efficient method of carbon fixation is a unique adaptation that combines close vein spacing and distinct photosynthetic cell types. Despite its importance, the molecular genetic basis of C4 leaf development is still poorly understood. Here we summarize current knowledge of leaf venation and review recent progress in understanding molecular and genetic regulation of vascular patterning events in C4 plants. Evidence points to the interplay of auxin, brassinosteroids, SHORTROOT/SCARECROW and INDETERMINATE DOMAIN transcription factors. Identification and functional characterization of candidate regulators acting early in vascular development will be essential for further progress in understanding the precise regulation of these processes.

Verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes.

MOTIVATION: Chloroplast genomes are now produced in the hundreds for angiosperm phylogenetics projects, but current methods for annotation, alignment and tree estimation still require some manual intervention reducing throughput and increasing analysis time for large chloroplast systematics projects. RESULTS: Verdant is a web-based software suite and database built to take advantage a novel annotation program, annoBTD. Using annoBTD, Verdant provides accurate annotation of chloroplast genomes without manual intervention. Subsequent alignment and tree estimation can incorporate newly annotated and publically available plastomes and can accommodate a large number of taxa. Verdant sharply reduces the time required for analysis of assembled chloroplast genomes and removes the need for pipelines and software on personal hardware. AVAILABILITY AND IMPLEMENTATION: Verdant is available at: http://verdant.iplantcollaborative.org/plastidDB/ It is implemented in PHP, Perl, MySQL, Javascript, HTML and CSS with all major browsers supported. CONTACT: mrmckain@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.

Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function.

CLV3/ESR (CLE) proteins are important signaling peptides in plants. The short CLE peptide (12-13 amino acids) is cleaved from a larger pre-propeptide and functions as an extracellular ligand. The CLE family is large and has resisted attempts at classification because the CLE domain is too short for reliable phylogenetic analysis and the pre-propeptide is too variable. We used a model-based search for CLE domains from 57 plant genomes and used the entire pre-propeptide for comprehensive clustering analysis. In total, 1628 CLE genes were identified in land plants, with none recognizable from green algae. These CLEs form 12 groups within which CLE domains are largely conserved and pre-propeptides can be aligned. Most clusters contain sequences from monocots, eudicots and Amborella trichopoda, with sequences from Picea abies, Selaginella moellendorffii and Physcomitrella patens scattered in some clusters. We easily identified previously known clusters involved in vascular differentiation and nodulation. In addition, we found a number of discrete groups whose function remains poorly characterized. Available data indicate that CLE proteins within a cluster are likely to share function, whereas those from different clusters play at least partially different roles. Our analysis provides a foundation for future evolutionary and functional studies.

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

Cross species selection scans identify components of C4 photosynthesis in the grasses.

C4 photosynthesis is perhaps one of the best examples of convergent adaptive evolution with over 25 independent origins in the grasses (Poaceae) alone. The availability of high quality grass genome sequences presents new opportunities to explore the mechanisms underlying this complex trait using evolutionary biology-based approaches. In this study, we performed genome-wide cross-species selection scans in C4 lineages to facilitate discovery of C4 genes. The study was enabled by the well conserved collinearity of grass genomes and the recently sequenced genome of a C3 panicoid grass, Dichanthelium oligosanthes This method, in contrast to previous studies, does not rely on any a priori knowledge of the genes that contribute to biochemical or anatomical innovations associated with C4 photosynthesis. We identified a list of 88 candidate genes that include both known and potentially novel components of the C4 pathway. This set includes the carbon shuttle enzymes pyruvate, phosphate dikinase, phosphoenolpyruvate carboxylase and NADP malic enzyme as well as several predicted transporter proteins that likely play an essential role in promoting the flux of metabolites between the bundle sheath and mesophyll cells. Importantly, this approach demonstrates the application of fundamental molecular evolution principles to dissect the genetic basis of a complex photosynthetic adaptation in plants. Furthermore, we demonstrate how the output of the selection scans can be combined with expression data to provide additional power to prioritize candidate gene lists and suggest novel opportunities for pathway engineering.

Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae.

Background and Aims: Independent evolution of derived complex characters provides a unique opportunity to assess whether and how similar genetic changes correlate with morphological convergence. Bilaterally symmetrical corollas have evolved multiple times independently from radially symmetrical ancestors and likely represent adaptations to attract specific pollinators. On the other hand, losses of bilateral corolla symmetry have occurred sporadically in various groups, due to either modification of bilaterally symmetrical corollas in late development or early establishment of radial symmetry. Methods: This study integrated phylogenetic, scanning electron microscopy (SEM)-based morphological, and gene expression approaches to assess the possible mechanisms underlying independent evolutionary losses of corolla bilateral symmetry. Key Results: This work compared three species of Lamiaceae having radially symmetrical mature corollas with a representative sister taxon having bilaterally symmetrical corollas and found that each reaches radial symmetry in a different way. Higher core Lamiales share a common duplication in the CYCLOIDEA (CYC ) 2 gene lineage and show conserved and asymmetrical expression of CYC2 clade and RAD genes along the adaxial-abaxial floral axis in species having bilateral corolla symmetry. In Lycopus americanus , the development and expression pattern of La-CYC2A and La-CYC2B are similar to those of their bilaterally symmetrical relatives, whereas the loss of La-RAD expression correlates with a late switch to radial corolla symmetry. In Mentha longifolia , late radial symmetry may be explained by the loss of Ml-CYC2A , and by altered expression of two Ml-CYC2B and Ml-RAD genes . Finally, expanded expression of Cc-CYC2A and Cc-RAD strongly correlates with the early development of radially symmetrical corollas in Callicarpa cathayana . Conclusions: Repeated losses of mature corolla bilateral symmetry in Lamiaceae are not uncommon, and may be achieved by distinct mechanisms and various changes to symmetry genes, including the loss of a CYC2 clade gene from the genome, and/or contraction, expansion or alteration of CYC2 clade and RAD -like gene expression.