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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.

Cotton fibers develop from epidermal cells in the outer integument of ovules. The regulatory process underlying fiber cell development has been extensively studied, little is known about the spatio-temporal profiles of transcriptomes and metabolomes during its development. Here we characterized the dynamics of transcriptome and metabolome during the early developmental stages of cotton fiber cells using a combination of spatial transcriptomic, single-cell transcriptomic, and spatial metabolomic analyses. We identified the key genetic regulators and metabolites that initiate and determine the fate of fiber cells. Both knockdown and gain-of-function analyses of one new identified marker gene BEE3/Gh_A09G062900 revealed its important role in the initiation of cotton fiber. We also designed a website for the public visualization of spatial gene expression in cotton that provides important reference datasets of spatio-temporal gene expression and can be used to further decipher the genetic and metabolic regulation of cotton fiber development (http://CottonOvule.cricaas.com.cn/).

Somatic embryogenesis is a major pathway for the regeneration of cotton in tissue culture, and a major technique used for cotton breeding and improvement. The signaling pathway regulating the process of somatic embryogenesis in cotton are extremely complex and still poorly characterized. Little is known about the mechanisms regulating the changes that occur in gene expression and metabolites during somatic embryogenesis at a single-cell resolution. Here, we investigated the spatial and single-cell expression profiles of key genes and metabolic patterns of key metabolites by integrated single-cell RNA-sequencing, spatial transcriptomics, and spatial metabolomics. To evaluate the results of the above analyses, we characterized the potential roles of two representative marker genes AATP1 and DOX2 in the regulation of somatic embryo development. A publicly accessible web-based resource database (http://cottonsomaticembryo.cricaas.com.cn) was generated to facilitate future studies designed to analyze the expression patterns of marker genes at specific developmental stages.

Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes ( PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase β-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in seedlings affected in AT5G08670, the gene of the β-subunit of mitochondrial ATP synthase than in wild-type (WT) seedlings upon treatment with lincomycin (LIN) and norflurazon (NF). Further studies showed that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in AT5G08670 mutant seedlings treated with LIN. These changes might be associated with the repression of some transcriptional factors (TFs), such as ARF3/ETTIN ( ETT), PLASTID TRANSCRIPTION FACTOR 1 ( PTF1), CYCLOIDEA AND PCF TRANSCRIPTION FACTOR 2 ( TCP2), and KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 ( KTF1). These findings indicate that the activity of mitochondrial ATP synthase affects plastid retrograde signaling.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants towards both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid (DAA) and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 ( ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE ( PIP), PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5 ( PRS5), and NITRILASE 3 ( NIT3). Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

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.

Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells (PCs), guard cells, and trichomes cells (TCs), which differentiate from protodermal cells or meristemoids. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid (JA), and transcription factors. Understanding 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 its 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 the bZIP25 and bZIP53, which are highly expressed in PCs and early-stage meristemoid cells. Densities of PCs and TCs 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 JA, suggesting that these transcription factors regulate the development of TCs and PCs in response to JA.IN A NUTSHELLBackgroundLeaf epidermal cells, comprised of trichome cells (TCs), guard cells (GCs), and pavement cells (PCs), are responsible for exchanging materials and information between plants and the surrounding aerial environment. Many genes have been identified in Arabidopsis thaliana and confirmed to be involved in the initiation and differentiation of TCs and PCs. The fate determination of TCs and PCs is tightly regulated by positive and negative regulators at the cellular level. The precise underlying molecular mechanisms responsible for the fate determination of TCs and PCs, however, are still unclear at this time.QuestionWhat are the transcriptomic profiles of different leaf epidermal cell types? Can we dissect the genes that are specifically expressed in certain epidermal cell types? What kinds of transcription factors are involved in regulating the fate determination of TCs and PCs?FindingsWe performed single cell RNA-seq to investigate the transcriptomic profiles of different leaf epidermal cell types and identified differentially expressed genes in each cell type. We found that genes that are involved in jasmonic acid signaling are highly expressed in early-stage meristemoid (EM) cells which can act as the precursor of PCs and perhaps of TCs. To investigate the regulatory mechanisms underlying EM development, we identified the transcription factors (TFs) in EM cells and found that two bZIP TF genes, bZIP25 and bZIP53, are highly expressed in EMs. Further analyses of these two genes using both loss-of-function and gain-of-function approaches indicated that bZIP25 and bZIP53 are functionally involved in promoting trichome formation but inhibit pavement cell development in response to jasmonic acid.Next stepsBesides of bZIP25 and bZIP53, we also identified other key genes, for example FES1B, in leaf epidermal cells. Our next step will be to explore the regulation of other key genes involved in the fate determination of different cell types in leaf epidermis.

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One new genus and four new species of the tribe Tegonotini (Eriophyidae: Phyllocoptinae) from Guangxi Zhuang Autonomous Region, China are described and illustrated: Asetidicrothrix luculiae gen. nov. and sp. nov. infesting Luculia sp. (Rubiaceae), Phyllocoptacus aporusae sp. nov. infesting Aporusa chinensis (Champ.) Merr. (Euphorbiaceae), Shevtchenkella acer sp. nov. infesting Acer davidii Franch. (Aceraceae) and Tegophyes embelia sp. nov. infesting Embelia oblongifolia Hemsl. (Myrsinaceae). All species described here are vagrants on the undersurface of host leaves. A key to the genera of Tegonotini from China is provided.

Four new species in the genus Diptacus from Guangxi Zhuang Autonomous Region, South China are described and illustrated in this paper: Diptacus actinodaphne Wang and Wei, n. sp. infesting Actinodaphne acuminata Meissn (Lauraeceae); Diptacus alniphyllus Wei, Wang and Li, n. sp. infesting Alniphyllum fortunei (Hemsl.) Makino (Styracaceae); Diptacus bracteatus Li, Wei and Qin, n. sp. infesting Vaccinium bracteatum Thunb. (Eriaceae); and Diptacus rhamnus Wei, Wang and Qin, n. sp. infesting Rhamnus crenata Decne (Rhamnaceae). A key to the species of Diptacus from China is provided.