Identification of bZIP Transcription Factors That Regulate the Development of Leaf Epidermal Cells in Arabidopsis thaliana by Single-Cell RNA Sequencing.

Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells, guard cells, and trichome cells. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid, and transcription factors. How the fate of leaf epidermal cells is determined, however, is still largely unknown due to the diversity of cell types and the complexity of their regulation. Here, we characterized the transcriptional profiles of epidermal cells in 3-day-old true leaves of Arabidopsis thaliana using single-cell RNA sequencing. We identified two genes encoding BASIC LEUCINE-ZIPPER (bZIP) transcription factors, namely bZIP25 and bZIP53, which are highly expressed in pavement cells and early-stage meristemoid cells. Densities of pavement cells and trichome cells were found to increase and decrease, respectively, in bzip25 and bzip53 mutants, compared with wild-type plants. This trend was more pronounced in the presence of jasmonic acid, suggesting that these transcription factors regulate the development of trichome cells and pavement cells in response to jasmonic acid.

Identification of novel regulators required for early development of vein pattern in the cotyledons by single-cell RNA-sequencing.

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

Research strategies for single-cell transcriptome analysis in plant leaves.

The recent and continuous improvement in single-cell RNA sequencing (scRNA-seq) technology has led to its emergence as an efficient experimental approach in plant research. However, compared with single-cell research in animals and humans, the application of scRNA-seq in plant research is limited by several challenges, including cell separation, cell type annotation, cellular function analysis, and cell-cell communication networks. In addition, the unavailability of corresponding reliable and stable analysis methods and standards has resulted in the relative decentralization of plant single-cell research. Considering these shortcomings, this review summarizes the research progress in plant leaf using scRNA-seq. In addition, it describes the corresponding feasible analytical methods and associated difficulties and problems encountered in the current research. In the end, we provide a speculative overview of the development of plant single-cell transcriptome research in the future.

Genetic regulation of nitrogen use efficiency in Gossypium spp.

Cotton (Gossypium spp.) is the most important fibre crop, with desirable characteristics preferred for textile production. Cotton fibre output relies heavily on nitrate as the most important source of inorganic nitrogen (N). However, nitrogen dynamics in extreme environments limit plant growth and lead to yield loss and pollution. Therefore, nitrogen use efficiency (NUE), which involves the utilisation of the 'right rate', 'right source', 'right time', and 'right place' (4Rs), is key for efficient N management. Recent omics techniques have genetically improved NUE in crops. We herein highlight the mechanisms of N uptake and assimilation in the vegetative and reproductive branches of the cotton plant while considering the known and unknown regulatory factors. The phylogenetic relationships among N transporters in four Gossypium spp. have been reviewed. Further, the N regulatory genes that participate in xylem transport and phloem loading are also discussed. In addition, the functions of microRNAs and transcription factors in modulating the expression of target N regulatory genes are highlighted. Overall, this review provides a detailed perspective on the complex N regulatory mechanism in cotton, which would accelerate the research toward improving NUE in crops.

Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development.

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis.

Molecular Regulation of Cotton Fiber Development: A Review.

Cotton (Gossypium spp.) is an economically important natural fiber crop. The quality of cotton fiber has a substantial effect on the quality of cotton textiles. The identification of cotton fiber development-related genes and exploration of their biological functions will not only enhance our understanding of the elongation and developmental mechanisms of cotton fibers but also provide insights that could aid the cultivation of new cotton varieties with improved fiber quality. Cotton fibers are single cells that have been differentiated from the ovule epidermis and serve as a model system for research on single-cell differentiation, growth, and fiber production. Genes and fiber formation mechanisms are examined in this review to shed new light on how important phytohormones, transcription factors, proteins, and genes linked to fiber development work together. Plant hormones, which occur in low quantities, play a critically important role in regulating cotton fiber development. Here, we review recent research that has greatly contributed to our understanding of the roles of different phytohormones in fiber development and regulation. We discuss the mechanisms by which phytohormones regulate the initiation and elongation of fiber cells in cotton, as well as the identification of genes involved in hormone biosynthetic and signaling pathways that regulate the initiation, elongation, and development of cotton fibers.

Single-Cell RNA Sequencing for Plant Research: Insights and Possible Benefits.

In recent years, advances in single-cell RNA sequencing (scRNA-seq) technologies have continued to change our views on biological systems by increasing the spatiotemporal resolution of our analysis to single-cell resolution. Application of scRNA-seq to plants enables the comprehensive characterization of both common and rare cell types and cell states, uncovering new cell types and revealing how cell types relate to each other spatially and developmentally. This review provides an overview of scRNA-seq methodologies, highlights the application of scRNA-seq in plant science, justifies why scRNA-seq is a master player of sequencing, and explains the role of single-cell transcriptomics technologies in environmental stress adaptation, alongside the challenges and prospects of single-cell transcriptomics. Collectively, we put forward a central role of single-cell sequencing in plant research.

An Overview of Cotton Gland Development and Its Transcriptional Regulation.

Cotton refers to species in the genus Gossypium that bear spinnable seed coat fibers. A total of 50 species in the genus Gossypium have been described to date. Of these, only four species, viz. Gossypium, hirsutum, G. barbadense, G. arboretum, and G. herbaceum are cultivated; the rest are wild. The black dot-like structures on the surfaces of cotton organs or tissues, such as the leaves, stem, calyx, bracts, and boll surface, are called gossypol glands or pigment glands, which store terpenoid aldehydes, including gossypol. The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that stores gossypol and its derivatives. It provides an ideal system for studying cell differentiation and organogenesis. However, only a few genes involved in the process of gland formation have been identified to date, and the molecular mechanisms underlying gland initiation remain unclear. The terpenoid aldehydes in the lysigenous glands of Gossypium species are important secondary phytoalexins (with gossypol being the most important) and one of the main defenses of plants against pests and diseases. Here, we review recent research on the development of gossypol glands in Gossypium species, the regulation of the terpenoid aldehyde biosynthesis pathway, discoveries from genetic engineering studies, and future research directions.

COE2 Is Required for the Root Foraging Response to Nitrogen Limitation.

There are numerous exchanges of signals and materials between leaves and roots, including nitrogen, which is one of the essential nutrients for plant growth and development. In this study we identified and characterized the Chlorophyll A/B-Binding Protein (CAB) (named coe2 for CAB overexpression 2) mutant, which is defective in the development of chloroplasts and roots under normal growth conditions. The phenotype of coe2 is caused by a mutation in the Nitric Oxide Associated (NOA1) gene that is implicated in a wide range of chloroplast functions including the regulation of metabolism and signaling of nitric oxide (NO). A transcriptome analysis reveals that expression of genes involved in metabolism and lateral root development are strongly altered in coe2 seedlings compared with WT. COE2 is expressed in hypocotyls, roots, root hairs, and root caps. Both the accumulation of NO and the growth of lateral roots are enhanced in WT but not in coe2 under nitrogen limitation. These new findings suggest that COE2-dependent signaling not only coordinates gene expression but also promotes chloroplast development and function by modulating root development and absorption of nitrogen compounds.

FLS2-RBOHD-PIF4 Module Regulates Plant Response to Drought and Salt Stress.

As sessile organisms, plants are constantly challenged by several environmental stresses. Different kinds of stress often occur simultaneously, leading to the accumulation of reactive oxygen species (ROS) produced by respiratory burst oxidase homolog (RBOHD) and calcium fluctuation in cells. Extensive studies have revealed that flagellin sensitive 2 (FLS2) can sense the infection by pathogenic microorganisms and activate cellular immune response by regulating intracellular ROS and calcium signals, which can also be activated during plant response to abiotic stress. However, little is known about the roles of FLS2 and RBOHD in regulating abiotic stress. In this study, we found that although the fls2 mutant showed tolerance, the double mutant rbohd rbohf displayed hypersensitivity to abiotic stress, similar to its performance in response to immune stress. An analysis of the transcriptome of the fls2 mutant and rbohd rbohf double mutant revealed that phytochrome interacting factor 4 (PIF4) acted downstream of FLS2 and RBOHD to respond to the abiotic stress. Further analysis showed that both FLS2 and RBOHD regulated the response of plants to drought and salt stress by regulating the expression of PIF4. These findings revealed an FLS2-RBOHD-PIF4 module in regulating plant response to biotic and abiotic stresses.

Identification of the Regulators of Epidermis Development under Drought- and Salt-Stressed Conditions by Single-Cell RNA-Seq.

As sessile organisms, plants constantly face challenges from the external environment. In order to meet these challenges and survive, plants have evolved a set of sophisticated adaptation strategies, including changes in leaf morphology and epidermal cell development. These developmental patterns are regulated by both light and hormonal signaling pathways. However, our mechanistic understanding of the role of these signaling pathways in regulating plant response to environmental stress is still very limited. By applying single-cell RNA-Seq, we determined the expression pattern of PHYTOCHROME INTERACTING FACTOR (PIF) 1, PIF3, PIF4, and PIF5 genes in leaf epidermal pavement cells (PCs) and guard cells (GCs). PCs and GCs are very sensitive to environmental stress, and our previous research suggests that these PIFs may be involved in regulating the development of PCs, GCs, and leaf morphology under environmental stress. Growth analysis showed that pif1/3/4/5 quadruple mutant maintained tolerance to drought and salt stress, and the length to width ratio of leaves and petiole length under normal growth conditions were similar to those of wild-type (WT) plants under drought and salt treatment. Analysis of the developmental patterns of PCs and GCs, and whole leaf morphology, further confirmed that these PIFs may be involved in mediating the development of epidermal cells under drought and salt stress, likely by regulating the expression of MUTE and TOO MANY MOUTHS (TMM) genes. These results provide new insights into the molecular mechanism of plant adaptation to adverse growth environments.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions.

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 µmol photons m-2s-1) compared to normal light (100 µmol photons m-2s-1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

COE 1 and GUN1 regulate the adaptation of plants to high light stress.

In order to withstand high light (HL) stress, plants have evolved both short-term defense and repair mechanisms and long-term acclimation responses. At present, however, the underlying signaling events and molecular mechanisms are still poorly understood. Analysis of the mutants coe1, coe1 gun1 double mutant and oeGUN1coe1 revealed increased sensitivity to HL stress as compared to wild type (WT), with oeGUN1 coe1 plants displaying the highest sensitivity. Accumulation of FTSH2 protein and degradation of D1 protein during the HL stress were shown to depend on both COE1 and GUN1. Overexpression of COE1 enhanced the induction of FTSH2 and the tolerance to HL stress. These results indicate that the COE1-GUN1 signaling pathway plays an important role in regulating the adaptation of plants to HL.

WRKY33-PIF4 loop is required for the regulation of H2O2 homeostasis.

The reactive oxygen species (ROS) are continuously produced and are essential for mediating the growth and development of plants. However too much accumulation of ROS can result in the oxidative damage to cells, especially under the adverse environmental conditions. Plants have evolved sophisticated strategies to regulate the homeostasis of H2O2. In this study, we generated transgenic Arabidopsis plants in the Ws ecotype (Ws) background in which WRKY33 is co-suppressed (csWRKY33/Ws). Compared with Ws, csWRKY33/Ws plants accumulate more H2O2. RNA-seq analysis indicated that in csWRKY33/Ws plants, expression of oxidative stress related genes such as ascorbate peroxidase 2 (APX2) is affected. Over-expression of APX2 can rescue the phenotype of csWRKY33/Ws, suggesting that the changes in the growth of csWRKY33/Ws is duo to the higher accumulation of H2O2. Analysis of the CHIP-seq data suggested that WRKY33 can directly regulate the expression of PIF4, vice versa. qPCR analysis also confirmed that the mutual regulation between WRKY33 and PIF4. Similar to that of csWRKY33/Ws, and the accumulation of H2O2 in pif4 also increased. Taken together, our results reveal a WRKY33-PIF4 regulatory loop that appears to play an important role in regulating the growth and development of seedlings by mediating H2O2 homeostasis.

Profiling of the Receptor for Activated C Kinase 1a (RACK1a) interaction network in Arabidopsis thaliana.

As a scaffold protein, Receptor for Activated C Kinase 1a (RACK1) interacts with many proteins and is involved in multiple biological processes in Arabidopsis. However, the global RACK1 protein interaction network in higher plants remains poorly understood. Here, we generated a yeast two-hybrid library using mixed samples from different developmental stages of Arabidopsis thaliana. Using RACK1a as bait, we performed a comprehensive screening of the resulting library to identify RACK1a interactors at the whole-transcriptome level. We selected 1065 independent positive clones that led to the identification of 215 RACK1a interactors. We classified these interactors into six groups according to their potential functions. Several interactors were selected for bimolecular fluorescence complementation (BiFC) analysis and their interaction with RACK1a was confirmed in vivo. Our results provide further insight into the molecular mechanisms through which RACK1a regulates various growth and development processes in higher plants.

DEG9, a serine protease, modulates cytokinin and light signaling by regulating the level of ARABIDOPSIS RESPONSE REGULATOR 4.

Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components.

Chloroplast retrograde signal regulates flowering.

Light is a major environmental factor regulating flowering time, thus ensuring reproductive success of higher plants. In contrast to our detailed understanding of light quality and photoperiod mechanisms involved, the molecular basis underlying high light-promoted flowering remains elusive. Here we show that, in Arabidopsis, a chloroplast-derived signal is critical for high light-regulated flowering mediated by the FLOWERING LOCUS C (FLC). We also demonstrate that PTM, a PHD transcription factor involved in chloroplast retrograde signaling, perceives such a signal and mediates transcriptional repression of FLC through recruitment of FVE, a component of the histone deacetylase complex. Thus, our data suggest that chloroplasts function as essential sensors of high light to regulate flowering and adaptive responses by triggering nuclear transcriptional changes at the chromatin level.

BASIC PENTACYSTEINE Proteins Repress ABSCISIC ACID INSENSITIVE4 Expression via Direct Recruitment of the Polycomb-Repressive Complex 2 in Arabidopsis Root Development.

Plant transcription factors generally act in complex regulatory networks that function at multiple levels to govern plant developmental programs. Dissection of the interconnections among different classes of transcription factors can elucidate these regulatory networks and thus improve our understanding of plant development. Here, we investigated the molecular and functional relationships of the transcription factors ABSCISIC ACID INSENSITIVE 4 (ABI4) and members of the BASIC PENTACYSTEINE (BPC) family in lateral root (LR) development of Arabidopsis thaliana. Genetic analysis showed that BPCs promote LR development by repressing ABI4 expression. Molecular analysis showed that BPCs bind to the ABI4 promoter and repress ABI4 transcription in roots. BPCs directly recruit the Polycomb Repressive Complex 2 (PRC2) to the ABI4 locus and epigenetically repress ABI4 expression by catalyzing the trimethylation of histone H3 at Lys27. In addition, BPCs and ABI4 co-ordinate their activities to fine-tune the levels of PIN-FORMED1, a component of the auxin signaling pathway, and thus modulate LR formation. These results establish a functional relationship between two universal and multiple-role transcription factors, and provide insight into the mechanisms of the transcriptional regulatory networks that affect Arabidopsis organogenesis.

Site-specific nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis.

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.

Functional relationship between mTERF4 and GUN1 in retrograde signaling.

Plastid-to-nucleus retrograde signaling plays an important role in regulating the expression of photosynthesis-associated nuclear genes (PhANGs) in accordance with physiological demands on chloroplast biogenesis and function. Despite its fundamental importance, little is known about the molecular nature of the plastid gene expression (PGE)-dependent type of retrograde signaling. PGE is a multifaceted process, and several factors, including pentatricopeptide repeat (PPR) proteins, are involved in its regulation. The PPR protein GUN1 plays a central role in PGE-dependent retrograde signaling. In this study, we isolated a mutant exhibiting up-regulation of CHLOROPHYLL A/B-BINDING PROTEIN (CAB) under normal growth conditions (named coe1 for CAB overexpression 1). The coe1 mutant has a single-base mutation in the gene for mitochondrial transcription termination factor 4 (mTERF4)/BSM/RUG2, which plays a role in regulating the processing of certain plastid transcripts. Defects in GUN1 or mTERF4 de-repressed the expression of specific plastid mRNAs in the presence of lincomycin (LIN). In wild-type plants, treatment with LIN or spectinomycin (SPE) inhibited processing of plastid transcripts. Comparative analysis revealed that in gun1 and coe1/mterf4, but not in wild-type, gun4, or gun5 plants, the processing of plastid transcripts and expression levels of Lhcb1 mRNA were affected in opposite ways when plants were grown in the presence of LIN or SPE. In addition, the coe1 mutation affected the intracellular accumulation and distribution of GUN1, as well as its plastid signaling activity. Taken together, these results suggest that GUN1 and COE1 cooperate in PGE and retrograde signaling.