Decoding the gene regulatory network of endosperm differentiation in maize.

The persistent cereal endosperm constitutes the majority of the grain volume. Dissecting the gene regulatory network underlying cereal endosperm development will facilitate yield and quality improvement of cereal crops. Here, we use single-cell transcriptomics to analyze the developing maize (Zea mays) endosperm during cell differentiation. After obtaining transcriptomic data from 17,022 single cells, we identify 12 cell clusters corresponding to five endosperm cell types and revealing complex transcriptional heterogeneity. We delineate the temporal gene-expression pattern from 6 to 7 days after pollination. We profile the genomic DNA-binding sites of 161 transcription factors differentially expressed between cell clusters and constructed a gene regulatory network by combining the single-cell transcriptomic data with the direct DNA-binding profiles, identifying 181 regulons containing genes encoding transcription factors along with their high-confidence targets, Furthermore, we map the regulons to endosperm cell clusters, identify cell-cluster-specific essential regulators, and experimentally validated three predicted key regulators. This study provides a framework for understanding cereal endosperm development and function at single-cell resolution.

A maize epimerase modulates cell wall synthesis and glycosylation during stomatal morphogenesis.

The unique dumbbell-shape of grass guard cells (GCs) is controlled by their cell walls which enable their rapid responses to the environment. The molecular mechanisms regulating the synthesis and assembly of GC walls are as yet unknown. Here we have identified BZU3, a maize gene encoding UDP-glucose 4-epimerase that regulates the supply of UDP-glucose during GC wall synthesis. The BZU3 mutation leads to significant decreases in cellular UDP-glucose levels. Immunofluorescence intensities reporting levels of cellulose and mixed-linkage glucans are reduced in the GCs, resulting in impaired local wall thickening. BZU3 also catalyzes the epimerization of UDP-N-acetylgalactosamine to UDP-N-acetylglucosamine, and the BZU3 mutation affects N-glycosylation of proteins that may be involved in cell wall synthesis and signaling. Our results suggest that the spatiotemporal modulation of BZU3 plays a dual role in controlling cell wall synthesis and glycosylation via controlling UDP-glucose/N-acetylglucosamine homeostasis during stomatal morphogenesis. These findings provide insights into the mechanisms controlling formation of the unique morphology of grass stomata.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

The maize single-nucleus transcriptome comprehensively describes signaling networks governing movement and development of grass stomata.

The unique morphology of grass stomata enables rapid responses to environmental changes. Deciphering the basis for these responses is critical for improving food security. We have developed a planta platform of single-nucleus RNA-sequencing by combined fluorescence-activated nuclei flow sorting, and used it to identify cell types in mature and developing stomata from 33,098 nuclei of the maize epidermis-enriched tissues. Guard cells (GCs) and subsidiary cells (SCs) displayed differential expression of genes, besides those encoding transporters, involved in the abscisic acid, CO2, Ca2+, starch metabolism, and blue light signaling pathways, implicating coordinated signal integration in speedy stomatal responses, and of genes affecting cell wall plasticity, implying a more sophisticated relationship between GCs and SCs in stomatal development and dumbbell-shaped guard cell formation. The trajectory of stomatal development identified in young tissues, and by comparison to the bulk RNA-seq data of the MUTE defective mutant in stomatal development, confirmed known features, and shed light on key participants in stomatal development. Our study provides a valuable, comprehensive, and fundamental foundation for further insights into grass stomatal function.

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Application-based guidelines for best practices in plant flow cytometry.

Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.

Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis.

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.

Best practices in the flow cytometry of microalgae.

Microalgae are photosynthetic microorganisms with a major influence on global ecosystems. Further, owing to the production of various secondary metabolites, microalgae are also intensively studied for their enormous potential in biotechnology and its applications. While flow cytometry (FCM) is a fast and reliable method particularly suitable for genome size estimation in plant and animal studies, its application to microalgae often comes with many methodological challenges due to specific issues (e.g., cell wall composition, and presence of various secondary metabolites). Sample preparation requires considerable amounts of biomass, chemical fixation, and/or extraction of cellular components. In genome size estimation, appropriate methods for isolation of intact nuclei (using lysis buffers, razor-blade chopping, various enzymes, or bead-beating of cells) are essential for successful and high-quality analyses. Nuclear DNA amounts of microalgae diverge greatly, varying by almost 30,000-fold (0.01 to 286 pg). Even though new algal reference standards for genome size are now being introduced, animal red blood cells and nuclei from plant tissues are still predominantly used. Due to our limited knowledge of microalgal life cycles, particular caution should be taken during 1C/2C-value (or ploidy level) assignments.

Shapiro's Laws Revisited: Conventional and Unconventional Cytometry at CYTO2020.

Extracting relevant information from a very large excess of irrelevant debris. Judicious gating of PI-stained Arabidopsis leaf homogenates defines the position of a very minor proportion of nuclei within a two-dimensional frequency distribution, whose properties can then be finely dissected. From: Galbraith (2009). Cytometry Part A. © 2020 International Society for Advancement of Cytometry.

Flow Cytometry and Sorting in Arabidopsis.

Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in molecular biology, vol 1062. Humana Press, Totowa, pp 509-537], describes the application of techniques of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing (a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei, and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are emphasized.

Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.

Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.

Transcriptome analysis reveals key genes involved in the regulation of nicotine biosynthesis at early time points after topping in tobacco (Nicotiana tabacum L.).

BACKGROUND: Nicotiana tabacum is an important economic crop. Topping, a common agricultural practice employed with flue-cured tobacco, is designed to increase leaf nicotine contents by increasing nicotine biosynthesis in roots. Many genes are found to be differentially expressed in response to topping, particularly genes involved in nicotine biosynthesis, but comprehensive analyses of early transcriptional responses induced by topping are not yet available. To develop a detailed understanding of the mechanisms regulating nicotine biosynthesis after topping, we have sequenced the transcriptomes of Nicotiana tabacum roots at seven time points following topping. RESULTS: Differential expression analysis revealed that 4830 genes responded to topping across all time points. Amongst these, nine gene families involved in nicotine biosynthesis and two gene families involved in nicotine transport showed significant changes during the immediate 24 h period following topping. No obvious preference to the parental species was detected in the differentially expressed genes (DEGs). Significant changes in transcript levels of nine genes involved in nicotine biosynthesis and phytohormone signal transduction were validated by qRT-PCR assays. 549 genes encoding transcription factors (TFs), found to exhibit significant changes in gene expression after topping, formed 15 clusters based on similarities of their transcript level time-course profiles. 336 DEGs involved in phytohormone signal transduction, including genes functionally related to the phytohormones jasmonic acid, abscisic acid, auxin, ethylene, and gibberellin, were identified at the earliest time point after topping. CONCLUSIONS: Our research provides the first detailed analysis of the early transcriptional responses to topping in N. tabacum, and identifies excellent candidates for further detailed studies concerning the regulation of nicotine biosynthesis in tobacco roots.

The genome of Populus alba x Populus tremula var. glandulosa clone 84K.

Poplar 84K (Populus alba x P. tremula var. glandulosa) is a fast-growing poplar hybrid. Originated in South Korea, this hybrid has been extensively cultivated in northern China. Due to the economic and ecological importance of this hybrid and high transformability, we now report the de novo sequencing and assembly of a male individual of poplar 84K using PacBio and Hi-C technologies. The final reference nuclear genome (747.5 Mb) has a contig N50 size of 1.99 Mb and a scaffold N50 size of 19.6 Mb. Complete chloroplast and mitochondrial genomes were also assembled from the sequencing data. Based on similarities to the genomes of P. alba var. pyramidalis and P. tremula, we were able to identify two subgenomes, representing 356 Mb from P. alba (subgenome A) and 354 Mb from P. tremula var. glandulosa (subgenome G). The phased assembly allowed us to detect the transcriptional bias between the two subgenomes, and we found that the subgenome from P. tremula displayed dominant expression in both 84K and another widely used hybrid, P. tremula x P. alba. This high-quality poplar 84K genome will be a valuable resource for poplar breeding and for molecular biology studies.

BZU2/ZmMUTE controls symmetrical division of guard mother cell and specifies neighbor cell fate in maize.

Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (bizui2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Transient induction of a subset of ethylene biosynthesis genes is potentially involved in regulation of grapevine bud dormancy release.

KEY MESSAGE: Transient increases in ethylene biosynthesis, achieved by tight regulation of transcription of specific ACC oxidase and ACC synthase genes, play a role in activation of grapevine bud dormancy release. The molecular mechanisms regulating dormancy release in grapevine buds are as yet unclear. It has been hypothesized that its core involves perturbation of respiration which induces an interplay between ethylene and ABA metabolism that removes repression and allows regrowth. Roles for hypoxia and ABA metabolism in this process have been previously supported. The potential involvement of ethylene biosynthesis in regulation of dormancy release, which has received little attention so far, is now explored. Our results indicate that (1) ethylene biosynthesis is induced by hydrogen cyanamide (HC) and azide (AZ), known artificial stimuli of dormancy release, (2) inhibitors of ethylene biosynthesis and signalling antagonize dormancy release by HC/AZ treatments, (3) ethylene application induces dormancy release, (4) there are two sets of bud-expressed ethylene biosynthesis genes which are differentially regulated, (5) only one set is transiently upregulated by HC/AZ and during the natural dormancy cycle, concomitant with changes in ethylene levels, and (6) levels of ACC oxidase transcripts and ethylene sharply decrease during natural dormancy release, whereas ACC accumulates. Given these results, we propose that transient increases in ethylene biosynthesis prior to dormancy release, achieved primarily by regulation of transcription of specific ACC oxidase genes, play a role in activation of dormancy release.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Abscisic acid catabolism enhances dormancy release of grapevine buds.

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)-mediated repression of bud-meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up-regulation of VvA8H-CYP707A4, which encodes ABA 8'-hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H-CYP707A4 within grape buds, (b) the VvA8H-CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H-CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H-CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H-CYP707A4 level in the bud is up-regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA-mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.

Distinct gibberellin functions during and after grapevine bud dormancy release.

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It has been hypothesized that (i) abscisic acid (ABA) represses bud-meristem activity; (ii) perturbation of respiration induces an interplay between ethylene and ABA metabolism, which leads to removal of repression; and (iii) gibberellin (GA)-mediated growth is resumed. The first two hypothesis have been formally supported. The current study examines the third hypothesis regarding the potential involvement of GA in dormancy release. We found that during natural dormancy induction, levels of VvGA3ox, VvGA20ox, and VvGASA2 transcripts and of GA1 were decreased. However, during dormancy release, expression of these genes was enhanced, accompanied by decreased expression of the bud-expressed GA-deactivating VvGA2ox. Despite indications for its positive role during natural dormancy release, GA application had inhibitory effects on bud break. Hydrogen cyanamide up-regulated VvGA2ox and down-regulated VvGA3ox and VvGA20ox expression, reduced GA1 levels, and partially rescued the negative effect of GA. GA had an inhibitory effect only when applied simultaneously with bud-forcing initiation. Given these results, we hypothesize that during initial activation of the dormant bud meristem, the level of GA must be restricted, but after meristem activation an increase in its level serves to enhance primordia regrowth.