Single-cell analysis resolves the genetic program of primary vascular system formation in hybrid poplar

Despite the enormous potential of novel approaches to explore gene expression at a single-cell level, we lack a high-resolution and cell type-specific gene expression map of the shoot apex in woody perennials. We use single-nuclei RNA sequencing to determine the cell type-specific transcriptome of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of epidermal cells, leaf mesophyll, and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we created and applied a pipeline for interspecific single-cell expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of primary vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the basic principles underlying cell division and differentiation conserved between herbaceous and perennial species, which also allows the evaluation of the divergencies at single-cell resolution.

A Tissue-Specific Landscape of Alternative Polyadenylation, lncRNAs, TFs, and Gene Co-expression Networks in Liriodendron chinense

Liriodendron chinense is an economically and ecologically important deciduous tree species. Although the reference genome has been revealed, alternative polyadenylation (APA), transcription factors (TFs), long non-coding RNAs (lncRNAs), and co-expression networks of tissue-specific genes remain incompletely annotated. In this study, we used the bracts, petals, sepals, stamens, pistils, leaves, and shoot apex of L. chinense as materials for hybrid sequencing. On the one hand, we improved the annotation of the genome. We detected 13,139 novel genes, 7,527 lncRNAs, 1,791 TFs, and 6,721 genes with APA sites. On the other hand, we found that tissue-specific genes play a significant role in maintaining tissue characteristics. In total, 2,040 tissue-specific genes were identified, among which 9.2% of tissue-specific genes were affected by APA, and 1,809 tissue-specific genes were represented in seven specific co-expression modules. We also found that bract-specific hub genes were associated plant defense, leaf-specific hub genes were involved in energy metabolism. Moreover, we also found that a stamen-specific hub TF Lchi25777 may be involved in the determination of stamen identity, and a shoot-apex-specific hub TF Lchi05072 may participate in maintaining meristem characteristic. Our study provides a landscape of APA, lncRNAs, TFs, and tissue-specific gene co-expression networks in L. chinense that will improve genome annotation, strengthen our understanding of transcriptome complexity, and drive further research into the regulatory mechanisms of tissue-specific genes.

Cotranscriptional and Posttranscriptional Features of the Transcriptome in Soybean Shoot Apex and Leaf

Transcription is the first step of central dogma, in which the genetic information stored in DNA is copied into RNA. In addition to mature RNA sequencing (RNA-seq), high-throughput nascent RNA assays have been established and applied to provide detailed transcriptional information. Here, we present the profiling of nascent RNA from trifoliate leaves and shoot apices of soybean. In combination with nascent RNA (chromatin-bound RNA, CB RNA) and RNA-seq, we found that introns were largely spliced cotranscriptionally. Although alternative splicing (AS) was mainly determined at nascent RNA biogenesis, differential AS between the leaf and shoot apex at the mature RNA level did not correlate well with cotranscriptional differential AS. Overall, RNA abundance was moderately correlated between nascent RNA and mature RNA within each tissue, but the fold changes between the leaf and shoot apex were highly correlated. Thousands of novel transcripts (mainly non-coding RNA) were detected by CB RNA-seq, including the overlap of natural antisense RNA with two important genes controlling soybean reproductive development, FT2a and Dt1. Taken together, we demonstrated the adoption of CB RNA-seq in soybean, which may shed light on gene expression regulation of important agronomic traits in leguminous crops.

Systematic analyses of the MIR172 family members of Arabidopsis define their distinct roles in regulation of APETALA2 during floral transition

MicroRNAs (miRNAs) play important roles in regulating flowering and reproduction of angiosperms. Mature miRNAs are encoded by multiple MIRNA genes that can differ in their spatiotemporal activities and their contributions to gene regulatory networks, but the functions of individual MIRNA genes are poorly defined. We functionally analyzed the activity of all 5 Arabidopsis thaliana MIR172 genes, which encode miR172 and promote the floral transition by inhibiting the accumulation of APETALA2 (AP2) and APETALA2-LIKE (AP2-LIKE) transcription factors (TFs). Through genome editing and detailed confocal microscopy, we show that the activity of miR172 at the shoot apex is encoded by 3 MIR172 genes, is critical for floral transition of the shoot meristem under noninductive photoperiods, and reduces accumulation of AP2 and TARGET OF EAT2 (TOE2), an AP2-LIKE TF, at the shoot meristem. Utilizing the genetic resources generated here, we show that the promotion of flowering by miR172 is enhanced by the MADS-domain TF FRUITFULL, which may facilitate long-term silencing of AP2-LIKE transcription, and that their activities are partially coordinated by the TF SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 15. Thus, we present a genetic framework for the depletion of AP2 and AP2-LIKE TFs at the shoot apex during floral transition and demonstrate that this plays a central role in floral induction.

KNOX HOMOLOGS SHOOT MERISTEMLESS (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during embryonic meristem development in Arabidopsis thaliana

In Arabidopsis, the genes SHOOT MERISTEMLESS (STM) and CLAVATA3 (CLV3) antagonistically regulate shoot meristem development. STM is essential for both development and maintenance of the meristem, as stm mutants fail to develop a shoot meristem during embryogenesis. CLV3, on the other hand, negatively regulates meristem proliferation, and clv3 mutants possess an enlarged shoot meristem. Genetic interaction studies revealed that stm and clv3 dominantly suppress each other’s phenotypes. STM works in conjunction with its closely related homologue KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6) to promote meristem development and organ separation, as stm knat6 double mutants fail to form a meristem and produce a fused cotyledon. In this study, we show that clv3 fails to promote post-embryonic meristem formation in stm-1 background if we also remove KNAT6. stm-1 knat6 clv3 triple mutants result in early meristem termination and produce fused cotyledons similar to stm knat6 double mutant. Notably, the stm-1 knat6 and stm-1 knat6 clv3 alleles lack tissue in the presumed region of SAM. stm knat6 clv3 also showed reduced inflorescence size and shoot apex size as compared to clv3 single or stm clv3 double mutants. In contrast to previously published data, these data suggest that stm is epistatic to clv3 in postembryonic meristem development.HighlightSTM and KNAT6 genes determine post-embryonic meristem formation and activity in Arabidopsis. clv3 mutation is unable to rescue the stm knat6 meristemless phenotype.

Single-nucleus RNA-seq resolves spatiotemporal developmental trajectories in the tomato shoot apex

Single cell transcriptomics is revolutionizing our understanding of development and response to environmental cues1–3. Recent advances in single cell RNA sequencing (scRNA-seq) technology have enabled profiling gene expression pattern of heterogenous tissues and organs at single cellular level and have been widely applied in human and animal research4,5. Nevertheless, the existence of cell walls significantly encumbered its application in plant research. Protoplasts have been applied for scRNA-seq analysis, but mostly restricted to tissues amenable for wall digestion, such as root tips6–10. However, many cell types are resistant to protoplasting, and protoplasting may yield ectopic gene expression and bias proportions of cell types. Here we demonstrate a method with minimal artifacts for high-throughput single-nucleus RNA sequencing (snRNA-Seq) that we use to profile tomato shoot apex cells. The obtained high-resolution expression atlas identifies numerous distinct cell types covering major shoot tissues and developmental stages, delineates developmental trajectories of mesophyll cells, vasculature cells, epidermal cells, and trichome cells. In addition, we identify key developmental regulators and reveal their hierarchy. Collectively, this study demonstrates the power of snRNA-seq to plant research and provides an unprecedented spatiotemporal gene expression atlas of heterogeneous shoot cells.