Search for: All records

The abscission zone (AZ) consists of specialized cell layers where cell separation or breakage occurs that result in organ detachment. Microscopic observation of the AZ is crucial for understanding its function. The AZ undergoes cellular and physiological changes prior to abscission, such as cell death, loss of chlorophyll, and the production of reactive oxygen species (ROS). These changes can be visualized using specific dyes and indicators under light or fluorescent microscopes. However, one challenge of using these dyes is their inefficient penetration into the tissue, especially when the epidermal layer has thick secondary cell walls. In this chapter, a detailed protocol to overcome this challenge is described. Using the fruit AZ of Setaria viridis, in which the epidermal cell wall is thick and lignified, we gently fix the dissected tissue, embed it in the Cryomatrix, and trim off the outer cell layers using a cryostat. The tissue with exposed inner cells can then be stained with fluorescent dyes to visualize organelles of interest, or 3,3′-diaminobenzidine (DAB) to visualize hydrogen peroxide accumulated in the tissue. 

Cereal shattering and threshability, both involving disarticulation of grains from the mother plant, are important traits for cereal domestication and improvement. Recent studies highlighted diverse mechanisms influencing shattering and threshability, either through development of the disarticulation zone or floral structures enclosing or supporting the disarticulation unit. Differential lignification in the disarticulation zone is essential for rice shattering but not required for many other grasses. During shattering, the disarticulation zone undergoes either abscission leading to cell separation or cell breakage. Threshability can be affected by the morphology and toughness of the enclosing floral structures, and in some species, by the inherent weakness of the disarticulation zone. Fine-tuning shattering and threshability is essential for breeding wild and less domesticated cereals. 

Abstract 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. 

Abstract 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. 

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia , one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions. 

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 andSetaria. In rice,SH1inhibits lignification specifically in the AZ. However, the AZ ofSetariais nonlignified throughout, raising the question of howSH1functions in species without lignification.Crispr‐Cas9 knockout mutants ofSH1were generated inSetaria viridisand characterized with histology, cell wall and auxin immunofluorescence, transmission electron microscopy, hormonal treatment and RNA‐Seq analysis.Thesh1mutant lacks shattering, as expected. No differences in cell anatomy or cell wall components including lignin were observed betweensh1and 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 andsh1, with the signal of an antibody to auxin detected in thesh1chloroplast.SH1inSetariais required for activation of abscission through auxin signaling, which is not reported in other grass species.