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.

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.

Tissue-specific changes in the RNA structurome mediate salinity response in Arabidopsis.

Little is known concerning the effects of abiotic factors on in vivo RNA structures. We applied Structure-seq to assess the in vivo mRNA structuromes of Arabidopsis thaliana under salinity stress, which negatively impacts agriculture. Structure-seq utilizes dimethyl sulfate reactivity to identify As and Cs that lack base-pairing or protection. Salt stress refolded transcripts differentially in root versus shoot, evincing tissue specificity of the structurome. Both tissues exhibited an inverse correlation between salt stress-induced changes in transcript reactivity and changes in abundance, with stress-related mRNAs showing particular structural dynamism. This inverse correlation is more pronounced in mRNAs wherein the mean reactivity of the 5'UTR, CDS, and 3'UTR concertedly change under salinity stress, suggesting increased susceptibility to abundance control mechanisms in transcripts exhibiting this phenomenon, which we name "concordancy." Concordant salinity-induced increases in reactivity were notably observed in photosynthesis genes, thereby implicating mRNA structural loss in the well-known depression of photosynthesis by salt stress. Overall, changes in secondary structure appear to impact mRNA abundance, molding the functional specificity of the transcriptome under stress.

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.

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.

The G Protein ß-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement.

Heterotrimeric guanine nucleotide-binding (G) proteins are composed of Gα, Gß, and Gγ subunits and function as molecular switches in signal transduction. In Arabidopsis (Arabidopsis thaliana), there are one canonical Gα (GPA1), three extra-large Gα (XLG1, XLG2, and XLG3), one Gß (AGB1), and three Gγ (AGG1, AGG2, and AGG3) subunits. To elucidate AGB1 molecular signaling, we performed immunoprecipitation using plasma membrane-enriched proteins followed by mass spectrometry to identify the protein interactors of AGB1. After eliminating proteins present in the control immunoprecipitation, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. We identified all of the G protein subunits except XLG1, receptor-like kinases, Ca2+ signaling-related proteins, and 14-3-3-like proteins, all of which may couple with or modulate G protein signaling. We confirmed physical interaction between AGB1 and the receptor-like kinase FERONIA (FER) using bimolecular fluorescence complementation. The Rapid Alkalinization Factor (RALF) family of polypeptides have been shown to be ligands of FER. In this study, we demonstrate that RALF1 regulates stomatal apertures and does so in a G protein-dependent manner, inhibiting stomatal opening and promoting stomatal closure in Columbia but not in agb1 mutants. We further show that AGGs and XLGs, but not GPA1, participate in RALF1-mediated stomatal signaling. Our results suggest that FER acts as a G protein-coupled receptor for plant heterotrimeric G proteins.

Inter-relationships between the heterotrimeric Gß subunit AGB1, the receptor-like kinase FERONIA, and RALF1 in salinity response.

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor-like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G-protein-coupled-receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1-like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein ß subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt-induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na+ accumulation and decreasing K+ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1-independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling.

Heterotrimeric G proteins, consisting of Gα, Gß, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gßγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gßγ binding in yeast. We observed interaction of the XLGs with all three Gßγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gßγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.