Plant Cell Types Research Articles

Perspective: Transcriptomics of Cryopreserved Cells

Cryopreservation is a well-known strategy to conserve genetic resources at ultra-low temperature. However, there is still limited knowledge on the cellular processes and molecular adjustments that allow cells to withstand the multiple stresses to which they are exposed during cryopreservation. To evaluate these processes, transcriptomics, the sub-discipline of omics that simultaneously examines mRNA transcripts formed by transcription from the genome, has been recently used. This article reviews recent scientific studies which use the basic principles of cryopreservation practices together with transcriptomics approaches, within the conceptual framework of cryobiomics. Moreover, the connections between factors that may be useful to optimize and validate approaches for mammalian or plant cell cryopreservation are also assessed. Transcriptomic applications are mainly performed with methods such as reverse transcriptase polymerase chain reaction (RT-PCR), simultaneous polymerase chain reaction (real-time PCR), northern blot, microarray/biochip and gene expression analysis (SAGE). Transcriptomic technologies allow a global view of gene expression profiles of different mammalian or plant cell types to be obtained before and after cryopreservation under multiple stress conditions. For these processes, small amounts of RNA enable efficient transcriptomics analysis. Transcriptomic analysis of cryopreserved mammalian and plant cells provides a conceptual way to identify the genes and their relative alterations in transcriptional abundances together with non-coding RNAs involved in important pathways related to cell viability and proliferation during and after cryopreservation. Moreover, it greatly contributes to understanding of non-fatal cryodamage and related developmental disorders in cryopreserved mammalian oocytes and sperm. In addition, single cell transcriptomics has the potential to non-invasely monitor immune actions and to diagnose the stage of the inflammatory process in kidney. Finally, qRT-PCR and RNA-seq studies have also revealed that some transcription factors are effective at inducing cold tolerance in many plants by elevating the levels of soluble sugars, proline and unsaturated fatty acids in cells. Hence transcriptomics studies may also aid investigations of the main mechanisms behind the so-called 'cryo-recalcitrance' that is observed mostly in plant cells.

What Is a Plant Cell Type in the Age of Single-Cell Biology? It's Complicated.

One of the fundamental questions in developmental biology is how a cell is specified to differentiate as a specialized cell type. Traditionally, plant cell types were defined based on their function, location, morphology, and lineage. Currently, in the age of single-cell biology, researchers typically attempt to assign plant cells to cell types by clustering them based on their transcriptomes. However, because cells are dynamic entities that progress through the cell cycle and respond to signals, the transcriptome also reflects the state of the cell at a particular moment in time, raising questions about how to define a cell type. We suggest that these complexities and dynamics of cell states are of interest and further consider the roles signaling, stochasticity, cell cycle, and mechanical forces play in plant cell fate specification. Once established, cell identity must also be maintained. With the wealth of single-cell data coming out, the field is poised to elucidate both the complexity and dynamics of cell states.

Salinity-Induced Photorespiration in Populus Vascular Tissues Facilitate Nitrogen Reallocation.

Adaptation to abiotic stress is critical for the survival of perennial tree species. Salinity affects plant growth and productivity by interfering with major biosynthetic processes. Detrimental effects of salinity may vary between different plant tissues and cell types. However, spatial molecular mechanisms controlling plant responses to salinity stress are not yet thoroughly understood in perennial trees. We used laser capture microdissection in clones of Populus tremula x alba to isolate palisade and vascular cells of intermediary leaf from plants exposed to 150 mM NaCl for 10 days, followed by a recovery period. Cell-specific changes in proteins and metabolites were determined. Salinity induced a vascular-specific accumulation of proteins associated with photorespiration, and the accumulation of serine, 3-phosphoglycerate and NH4 + suggesting changes in N metabolism. Accumulation of the GLUTAMINE SYNTHETASE 2 protein, and increased GS1.1 gene expression, indicated that NH4 + produced in photorespiration was assimilated to glutamine, the main amino acid translocated in Populus trees. Further analysis of total soluble proteins in stems and roots showed the accumulation of bark storage proteins induced by the salinity treatments. Collectively, our results suggest that the salt-induced photorespiration in vascular cells mediates N-reallocation in Populus, an essential process for the adaptation of trees to adverse conditions.

Two Calcium Sensor-Activated Kinases Function in Root Hair Growth.

Plant pollen tubes and root hairs typical polarized tip growth. It is well established that calcium ions (Ca2+) play essential roles in maintaining cell polarity and guiding cell growth orientation. Ca2+ signals are encoded by Ca2+ channels and transporters and are decoded by a variety of Ca2+-binding proteins often called Ca2+ sensors, in which calcineurin B-like protein (CBL) proteins function by interacting with and activating a group of kinases, activate CBL-interacting protein kinases (CIPKs). Some CBL-CIPK complexes, such as CBL2/3-CIPK12/19, act as crucial regulators of pollen tube growth. Whether these calcium decoding components regulate the growth of root hairs, another type of plant cell featuring Ca2+-regulated polarized growth, remains unknown. In this study, we identified CIPK13 and CIPK18 as genes specifically expressed in Arabidopsis (Arabidopsis thaliana) root hairs. The cipk13 cipk18 double mutants showed reduced root hair length and lower growth rates. The calcium oscillations at the root hair tip were attenuated in the cipk13 cipk18 mutants as compared to the wild-type plants. Through yeast two-hybrid screens, CBL2 and CBL3 were identified as interacting with CIPK13 and CIPK18. cbl2 cbl3 displayed a shortened root hair phenotype similar to cipk13 cipk18. This genetic analysis, together with biochemical assays showing activation of CIPK13/18 by CBL2/3, supported the conclusion that CBL2/3 and CIPK13/18 may work as Ca2+-decoding modules in controlling root hair growth. Thus, the findings that CIPK12/19 and CIPK13/18 function in pollen tube and root hair growth, respectively, illustrate a molecular mechanism in which the same CBLs recruit distinct CIPKs in regulating polarized tip growth in different types of plant cells.

Single-cell proteomics differentiates Arabidopsis root cell types.

Single-cell proteomics (SCP) is an emerging approach to resolve cellular heterogeneity within complex tissues of multi-cellular organisms. Here, we demonstrate the feasibility of SCP on plant samples using the model plant Arabidopsis thaliana. Specifically, we focused on examining isolated single cells from the cortex and endodermis, which are two adjacent root cell types derived from a common stem cell lineage. From 756 root cells, we identified 3763 proteins and 1118 proteins/cell. Ultimately, we focus on 3217 proteins quantified following stringent filtering. Of these, we identified 596 proteins whose expression is enriched in either the cortex or endodermis and are able to differentiate these closely related plant cell types. Collectivity, this study demonstrates that SCP can resolve neighboring cell types with distinct functions, thereby facilitating the identification of biomarkers and candidate proteins to enable functional genomics.

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

Hydrogel Substrates of Collagen-Starch-Selenium Complexes for Stimulating Plant Metabolism

Various selenium (IV) complexes with essential amino acids such as phenylalanine, histidine, and tryptophan were encapsulated within polymeric matrices in hydrogel state based on collagen-starch, generating the biomatrices Col-St(Se-F), Col-St(Se-H), and Col-St(Se-T), respectively. 1 mg of each type of selenium complex was employed per mass ratio in matrices consisting of 60% collagen and 18% starch by mass. The advanced materials were surface-characterized using scanning electron microscopy (SEM), and their properties such as swelling capacity and degradation rate in commercial vegetable substrate were also evaluated. The amino acid involved in the selenium complex forms characteristic granules that determine the microstructure and porosity of the biomatrix; reporting that in the case of all biomatrices with selenium complexes, swelling capacities exceeding 2900% are shown, resulting statistically significant compared to the sole collagen-starch matrix. Likewise, the matrices exhibit resistance to degradation in the presence of the commercial vegetable substrate for up to 30 days of evaluation. In vitro biocompatibility studies with green and red tomato cells derived from fresh seeds reveal that the biomatrices Col-Str(Se-F) and Col-Str(Se-H) overstimulate the metabolism of green tomato cells, while for red tomato cells, greater stimulation is appreciated in the Col-Str(Se-T) biomatrix. The chemical composition of the biomatrices promotes the proliferation of these types of plant cells as inspected by epifluorescent microscopy. Such advanced materials could be successfully applied in agricultural applications related to tomato cultivation by regulating water capacity, substrate biodegradation, and stimulated metabolism to promote tomato growth.

The evolving definition of plant cell type.

The evolving definition of plant cell type.

Spatial regulation of plant hormone action.

Although many plant cell types are capable of producing hormones, and plant hormones can in most cases act in the same cells in which they are produced, they also act as signaling molecules that coordinate physiological responses between different parts of the plant, indicating that their action is subject to spatial regulation. Numerous publications have reported that all levels of plant hormonal pathways, namely metabolism, transport, and perception/signal transduction, can help determine the spatial ranges of hormone action. For example, polar auxin transport or localized auxin biosynthesis contribute to creating a differential hormone accumulation across tissues that is instrumental for specific growth and developmental responses. On the other hand, tissue specificity of cytokinin actions has been proposed to be regulated by mechanisms operating at the signaling stages. Here, we review and discuss current knowledge about the contribution of the three levels mentioned above in providing spatial specificity to plant hormone action. We also explore how new technological developments, such as plant hormone sensors based on FRET (fluorescence resonance energy transfer) or single-cell RNA-seq, can provide an unprecedented level of resolution in defining the spatial domains of plant hormone action and its dynamics.

A method for analyzing programmed cell death in xylem development by flow cytometry.

Programmed cell death (PCD) is a genetically regulated developmental process leading to the death of specific types of plant cells, which plays important roles in plant development and growth such as wood formation. However, an efficient method needs to be established to study PCD in woody plants. Flow cytometry is widely utilized to evaluate apoptosis in mammalian cells, while it is rarely used to detect PCD in plants, especially in woody plants. Here, we reported that the xylem cell protoplasts from poplar stem were stained with a combination of fluorescein annexin V-FITC and propidium iodide (PI) and then sorted by flow cytometry. As expected, living cells (annexin V-FITC negative/PI negative), early PCD cells (annexin V-FITC positive/PI negative), and late PCD cells (annexin V-FITC positive/PI positive) could be finely distinguished through this method and then subjected for quantitative analysis. The expression of cell-type- and developmental stages-specific marker genes was consistent with the cell morphological observation. Therefore, the newly developed fluorescence-activated cell sorting (FACS) method can be used to study PCD in woody plants, which will be beneficial for studying the molecular mechanisms of wood formation.

Phloem development.

The evolution of the plant vascular system is a key process in Earth history because it enabled plants to conquer land and transform the terrestrial surface. Among the vascular tissues, the phloem is particularly intriguing because of its complex functionality. In angiosperms, its principal components are the sieve elements, which transport phloem sap, and their neighboring companion cells. Together, they form a functional unit that sustains sap loading, transport, and unloading. The developmental trajectory of sieve elements is unique among plant cell types because it entails selective organelle degradation including enucleation. Meticulous analyses of primary, so-called protophloem in the Arabidopsis thaliana root meristem have revealed key steps in protophloem sieve element formation at single-cell resolution. A transcription factor cascade connects specification with differentiation and also orchestrates phloem pole patterning via noncell-autonomous action of sieve element-derived effectors. Reminiscent of vascular tissue patterning in secondary growth, these involve receptor kinase pathways, whose antagonists guide the progression of sieve element differentiation. Receptor kinase pathways may also safeguard phloem formation by maintaining the developmental plasticity of neighboring cell files. Our current understanding of protophloem development in the A. thaliana root has reached sufficient detail to instruct molecular-level investigation of phloem formation in other organs.

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The role of Atg16 in autophagy, anthocyanin biosynthesis, and programmed cell death in leaves of the lace plant (Aponogeton madagascariensis).

Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with "pre-perforation" furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the "window stage", anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained.

Tissue‐Specific Isolation of Tagged Arabidopsis Plastids

Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do not allow for tissue-specific resolution, which hinders our understanding of their physiology. Therefore, IPTACT (Isolation of Plastids TAgged in specific Cell Types) was developed to isolate plastids in a tissue-specific manner in Arabidopsis thaliana (Arabidopsis). Plastids are biotinylated using one-shot transgenic lines, and tissue specificity is achieved with a suitable promoter as long as such a promoter exists. Cell-specific biotinylated plastids are then isolated with 2.8-µm streptavidin beads. Plastids extracted by IPTACT are suitable for RNA or protein isolation and subsequent tissue-specific OMICs analyses. This method provides the user with a powerful tool to investigate plastidial functions at cell-type resolution. Furthermore, it can easily be combined with studies using diverse genetic backgrounds and/or different developmental or stress conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Promoter cloning and plant selection Basic Protocol 2: Isolation of biotinylated plastids Basic Protocol 3: Quality control of isolated plastids.

Long-term spatiotemporal and highly specific imaging of the plasma membrane of diverse plant cells using a near-infrared AIE probe.

Fluorescent probes are valuable tools to visualize plasma membranes intuitively and clearly and their related physiological processes in a spatiotemporal manner. However, most existing probes have only realized the specific staining of the plasma membranes of animal/human cells within a very short time period, while almost no fluorescent probes have been developed for the long-term imaging of the plasma membranes of plant cells. Herein, we designed an AIE-active probe with NIR emission to achieve four-dimensional spatiotemporal imaging of the plasma membranes of plant cells based on a collaboration approach involving multiple strategies, demonstrated long-term real-time monitoring of morphological changes of plasma membranes for the first time, and further proved its wide applicability to plant cells of different types and diverse plant species. In the design concept, three effective strategies including the similarity and intermiscibility principle, antipermeability strategy and strong electrostatic interactions were combined to allow the probe to specifically target and anchor the plasma membrane for an ultralong amount of time on the premise of guaranteeing its sufficiently high aqueous solubility. The designed APMem-1 can quickly penetrate cell walls to specifically stain the plasma membranes of all plant cells in a very short time with advanced features (ultrafast staining, wash-free, and desirable biocompatibility) and the probe shows excellent plasma membrane specificity without staining other areas of the cell in comparison to commercial FM dyes. The longest imaging time of APMem-1 can be up to 10 h with comparable performance in both imaging contrast and imaging integrity. The validation experiments on different types of plant cells and diverse plants convincingly proved the universality of APMem-1. The development of plasma membrane probes with four-dimensional spatial and ultralong-term imaging ability provides a valuable tool to monitor the dynamic processes of plasma membrane-related events in an intuitive and real-time manner.

ЦИТОТОКСИЧНА / ЦИТОСТАТИЧНА ДІЯ НА ПУХЛИННІ КЛІТИНИ ЛІНІЇ HELA КУЛЬТИВОВАНИХ ГРИБІВ FLAMMULINA VELUTIPES, CYCLOCYBE AEGERITA ТА HERICIUM ERINACEUS

The use in clinical and pharmaceutical practice of biological preparations obtained from natural raw materials, in particular from mushrooms, is of urgent importance. Currently, the cultivation technologies of medicinal mushrooms are widely used, the mycelium of which contains a significant amount of cytokinins – stimulators of proliferation and differentiation of various types of plant cells, which also exhibit an immunomodulatory and oncostatic effects in the organism of animals and humans. Therefore, the aim of this work was to determine the cytotoxic/cytostatic effect of cultivated mushrooms Flammulina velutipes, Cyclocybe aegerita and Hericium erinaceus. Purification of cytokinins was carried out in stages, using methods of centrifugation, fractionation with n-butanol, ion exchange chromatography on Dowex 50Wx8 columns and thin-layer chromatography on Silicagel 60 F254. Identification and quantification of cytokinins was performed by by liquid chromatography in the reverse phase of MS (Agilent 1200 LC, USA) on an Agilent Zorbax Eclipse XDB-C18 column. Cytotoxic/cytostatic screening was performed on HeLa cells derived from human cervical cancer tumors using the MTT assay and routine counting of the ratio of live to dead cells. The results of the research showed that the highest content of cytokinins per 1 g of dry weight was characteristic of cultivated mushrooms of the species H. erinaceus. Also, H. erinaceus has the highest toxicity against human cervical cancer cells of the HeLa line, while the mushrooms F. velutipes and C. aegerita showed a cytostatic (antiproliferative) effect.

Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

Transcriptome profiling of celery petiole tissues reveals peculiarities of the collenchyma cell wall formation.

Transcriptome and biochemical analyses are applied to individual plant cell types to reveal potential players involved in the molecular machinery of cell wall formation in specialized cells such as collenchyma. Plant collenchyma is a mechanical tissue characterized by an irregular, thickened cell wall and the ability to support cell elongation. The composition of the collenchyma cell wall resembles that of the primary cell wall and includes cellulose, xyloglucan, and pectin; lignin is absent. Thus, the processes associated with the formation of the primary cell wall in the collenchyma can be more pronounced compared to other tissues due to its thickening. Primary cell walls intrinsic to different tissues may differ in structure and composition, which should be reflected at the transcriptomic level. For the first time, we conducted transcriptome profiling of collenchyma strands isolated from young celery petioles and compared them with other tissues, such as parenchyma and vascular bundles. Genes encoding proteins involved in the primary cell wall formation during cell elongation, such as xyloglucan endotransglucosylase/hydrolases, expansins, and leucine-rich repeat proteins, were significantly activated in the collenchyma. As the key players in the transcriptome orchestra of collenchyma, xyloglucan endotransglucosylase/hydrolase transcripts were characterized in more detail, including phylogeny and expression patterns. The comprehensive approach that included transcriptome and biochemical analyses allowed us to reveal peculiarities of collenchyma cell wall formation and modification, matching the abundance of upregulated transcripts and their potential substrates for revealed gene products. As a result, specific isoforms of multigene families were determined for further functional investigation.

Cell-Type-Specific Length and Cytosolic pH Response of Superficial Cells of Arabidopsis Root to Chronic Salinity.

Soil salinity negatively affects the growth, development and yield of plants. Acidification of the cytosol in cells of glycophytes was reported under salinity, while various types of plant cells can have a specific reaction under the same conditions. Transgenic Arabidopsis plants expressing the pH sensor Pt-GFP in the cytosol were used in this work for determination of morphometric changes and cytosolic pH changes in the superficial cells of Arabidopsis roots under chronic salinity in vitro. We did not find changes in the length of the root cap cells, while there was a decrease in the length of the differentiation zone under 50, 75 mM NaCl and the size of the epidermal cells of the differentiation zone under 75 mM NaCl. The most significant changes of cytosolic pH to chronic salinity was noted in columella (decrease by 1 pH unit at 75 mM NaCl) and epidermal cells of the differentiation zone (decrease by 0.6 and 0.4 pH units at 50 and 75 mM NaCl, respectively). In developed lateral root cap cells, acidification of cytosol by 0.4 units occurred only under 75 mM NaCl in the medium. In poorly differentiated lateral cells of the root cap, there were no changes in pH under chronic salinity.

MINI-EX: Integrative inference of single-cell gene regulatory networks in plants

Multicellular organisms, such as plants, are characterized by highly specialized and tightly regulated cell populations, establishing specific morphological structures and executing distinct functions. Gene regulatory networks (GRNs) describe condition-specific interactions of transcription factors (TFs) regulating the expression of target genes, underpinning these specific functions. As efficient and validated methods to identify cell-type-specific GRNs from single-cell data in plants are lacking, limiting our understanding of the organization of specific cell types in both model species and crops, we developed MINI-EX (Motif-Informed Network Inference based on single-cell EXpression data), an integrative approach to infer cell-type-specific networks in plants. MINI-EX uses single-cell transcriptomic data to define expression-based networks and integrates TF motif information to filter the inferred regulons, resulting in networks with increased accuracy. Next, regulons are assigned to different cell types, leveraging cell-specific expression, and candidate regulators are prioritized using network centrality measures, functional annotations, and expression specificity. This embedded prioritization strategy offers a unique and efficient means to unravel signaling cascades in specific cell types controlling a biological process of interest. We demonstrate the stability of MINI-EX toward input data sets with low number of cells and its robustness toward missing data, and show that it infers state-of-the-art networks with a better performance compared with other related single-cell network tools. MINI-EX successfully identifies key regulators controlling root development in Arabidopsis and rice, leaf development in Arabidopsis, and ear development in maize, enhancing our understanding of cell-type-specific regulation and unraveling the roles of different regulators controlling the development of specific cell types in plants.