From grasses to succulents - development and function of distinct stomatal subsidiary cells.

Stomata are breathing pores on leaves that balance photosynthetic carbon dioxide uptake and water vapor loss. Stomatal morphology and complexity are rather diverse when considering stomatal subsidiary cells (SCs). Subsidiary cells are adjacent to the central guard cells (GCs) and are morphologically distinct from other epidermal cells. Yet, how various SCs develop and whether and how they support stomatal gas exchange physiology outside of the grass family is largely unknown. Here, we discuss the development, ontogeny, and putative function of paracytic vs anisocytic SCs, which can be found in grasses and Crassulaceae succulents, respectively. First, we highlight recent advances in understanding how grasses form stomatal SCs. We then summarize novel insights into stomatal development in SC-less Arabidopsis to speculate on how this stomatal program might be rewired to enable anisocytic SC formation. Finally, we discuss the functional relevance of paracytic SCs in grasses and the putative roles of anisocytic SCs in succulents

Opposite polarity programs regulate asymmetric subsidiary cell divisions in grasses.

Grass stomata recruit lateral subsidiary cells (SCs), which are key to the unique stomatal morphology and the efficient plant-atmosphere gas exchange in grasses. Subsidiary mother cells (SMCs) strongly polarise before an asymmetric division forms a SC. Yet apart from a proximal polarity module that includes PANGLOSS1 (PAN1) and guides nuclear migration, little is known regarding the developmental processes that form SCs. Here, we used comparative transcriptomics of developing wild-type and SC-less bdmute leaves in the genetic model grass Brachypodium distachyon to identify novel factors involved in SC formation. This approach revealed BdPOLAR, which forms a novel, distal polarity domain in SMCs that is opposite to the proximal PAN1 domain. Both polarity domains are required for the formative SC division yet exhibit various roles in guiding pre-mitotic nuclear migration and SMC division plane orientation, respectively. Nonetheless, the domains are linked as the proximal domain controls polarisation of the distal domain. In summary, we identified two opposing polarity domains that coordinate the SC division, a process crucial for grass stomatal physiology

Consistent Reanalysis of Genome-wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets

Genomic imprinting leads to different expression levels of maternally and paternally derived alleles. Over the last years, major progress has been made in identifying novel imprinted candidate genes in plants, owing to affordable next-generation sequencing technologies. However, reports on sequencing the transcriptome of hybrid F1 seed tissues strongly disagree about how many and which genes are imprinted. This raises questions about the relative impact of biological, environmental, technical, and analytic differences or biases. Here, we adopt a statistical approach, frequently used in RNA-seq data analysis, which properly models count overdispersion and considers replicate information of reciprocal crosses. We show that our statistical pipeline outperforms other methods in identifying imprinted genes in simulated and real data. Accordingly, reanalysis of genome-wide imprinting studies in Arabidopsis and maize shows that, at least for Arabidopsis, an increased agreement across datasets could be observed. For maize, however, consistent reanalysis did not yield a larger overlap between the datasets. This suggests that the discrepancy across publications might be partially due to different analysis pipelines but that technical, biological, and environmental factors underlie much of the discrepancy between datasets. Finally, we show that the set of genes that can be characterized regarding allelic bias by all studies with minimal confidence is small (~8,000/27,416 genes for Arabidopsis and ~12,000/39,469 for maize). In conclusion, we propose to use biologically replicated reciprocal crosses, high sequence coverage, and a generalized linear model approach to identify differentially expressed alleles in developing seeds

Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays

The wild grass Brachypodium distachyon as a developmental model system

The arrival of cheap and high-throughput sequencing paired with efficient gene editing technologies allows us to use non-traditional model systems and mechanistically approach biological phenomena beyond what was conceivable just a decade ago. Venturing into different model systems enables us to explore for example clade-specific environmental responses to changing climates or the genetics and development of clade-specific organs, tissues and cell types. We—both early career researchers working with the wild grass model Brachypodium distachyon—want to use this review to (1) highlight why we think B. distachyon is a fantastic grass developmental model system, (2) summarize the tools and resources that have enabled discoveries made in B. distachyon, and (3) discuss a handful of developmental biology vignettes made possible by using B. distachyon as a model system. Finally, we want to conclude by (4) relating our personal stories with this emerging model system and (5) share what we think is important to consider before starting work with an emerging model system

Quantitative effects of environmental variation on stomatal anatomy and gas exchange in a grass model

Stomata are cellular pores on the leaf epidermis that allow plants to regulate carbon assimilation and water loss. Stomata integrate environmental signals to regulate pore apertures and adapt gas exchange to fluctuating conditions. Here, we quantified intraspecific plasticity of stomatal gas exchange and anatomy in response to seasonal variation in Brachypodium distachyon. Over the course of 2 years, we (a) used infrared gas analysis to assess light response kinetics of 120 Bd21-3 wild-type individuals in an environmentally fluctuating greenhouse and (b) microscopically determined the seasonal variability of stomatal anatomy in a subset of these plants. We observed systemic environmental effects on gas exchange measurements and remarkable intraspecific plasticity of stomatal anatomical traits. To reliably link anatomical variation to gas exchange, we adjusted anatomical g smax calculations for grass stomatal morphology. We propose that systemic effects and variability in stomatal anatomy should be accounted for in long-term gas exchange studies