Single cell RNA-sequencing (scRNA-seq) advancements have helped detect transcriptional heterogeneities in biological samples. However, scRNA-seq cannot currently provide high-resolution spatial transcriptome information or identify subcellular organs in biological samples. These limitations have led to the development of spatially enhanced-resolution omics-sequencing (Stereo-seq), which combines spatial information with single cell transcriptomics to address the challenges of scRNA-seq alone. In this review, we discuss the advantages of Stereo-seq technology. We anticipate that the application of such an integrated approach in plant research will advance our understanding of biological process in the plant transcriptomics era. We conclude with an outlook of how such integration will enhance crop improvement. 

Introducing single cell stereo-sequencing technology to transform the plant transcriptome landscape.

A spatial transcriptome map of the developing maize ear.

Leaf angle (LA) is a crucial factor affecting planting density and yield in maize. However, the regulatory mechanisms underlying LA formation remain largely unknown. In this study, we conducted a comparative histological analysis of the ligular region across various maize inbred lines, revealing that LA size is significantly influenced by a two-step regulatory process involving initial cell elongation followed by subsequent lignification in the ligular adaxial sclerenchyma cells (SC). We performed both bulk and single-nucleus RNA sequencing, generated a comprehensive transcriptomic atlas of the ligular region, and identified numerous genes enriched in the hypodermal cells that may influence their specialization into SC. Furthermore, we functionally characterized two genes encoding atypical basic helix-loop-helix (bHLH) transcription factors, bHLH30 and its homolog bHLH155, respectively, which are highly expressed in the elongated adaxial cells. Genetic analyses revealed that bHLH30 and bHLH155 positively regulate LA expansion, and molecular experiments demonstrated their ability to activate the transcription of genes involved in cell elongation and lignification of SC. These findings highlight the specialized functions of ligular adaxial SC in LA regulation by restricting the further extension of ligular cells and enhancing mechanical strength. The transcriptomic atlas of ligular region at single -nucleus resolution not only deepens our understanding of LA regulation but also identifies numerous potential targets for optimizing plant architecture in modern maize breeding. 

Histological and Single-nucleus Transcriptome Analyses Unveil the Specialized Functions of Ligular Sclerenchyma Cells and Key Regulators of Leaf Angle in Maize.

Abstract Plants possess remarkable capability to regenerate upon tissue damage or optimal environmental stimuli. This ability not only serves as a crucial strategy for immobile plants to survive through harsh environments, but also made numerous modern plant improvements techniques possible. At the cellular level, this biological process involves dynamic changes in gene expression that redirect cell fate transitions. It is increasingly recognized that chromatin epigenetic modifications, both activating and repressive, intricately interact to regulate this process. Moreover, the outcomes of epigenetic regulation on regeneration are influenced by factors such as the differences in regenerative plant species and donor tissue types, as well as the concentration and timing of hormone treatments. In this review, we focus on several well-characterized epigenetic modifications and their regulatory roles in the expression of widely studied morphogenic regulators, aiming to enhance our understanding of the mechanisms by which epigenetic modifications govern plant regeneration. 

The roles of epigenetic regulators in plant regeneration: exploring patterns amidst complex conditions

Over the past decade, bulk RNA sequencing (RNA-seq) has become an indispensable tool in molecular biology, and have made the novel development, with two innovative methodologies being developed, single-cell RNA sequencing (scRNA-seq) technology and spatial transcriptome (ST) technology. The scRNA-seq technology allows researchers to analyze gene expression in individual cells, providing more detailed information relative to the past technologies. Meanwhile, ST technology overcomes the limitations of single-cell sequencing in terms of loss of spatial information, enabling scientists to better understand the spatial distribution of gene expression within tissues. These advancements of transcriptomics technologies revolutionize the field of genomics and have been widely used in disease diagnosis and medicine. However, they are less utilized in plant research. This review describes the development, advantage and limitations of three transcriptomics technologies, and presents their applications in plant sciences. 

Development and application of transcriptomics technologies in plant science

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.


 
 
 Direct RNA-seq (dRNA-seq) using Oxford Nanopore Technology (ONT) has revolutionized transcript mapping by offering enhanced precision due to its long read length. Unlike traditional techniques, dRNA-seq eliminates the need for PCR amplification, reducing the impact of GC bias and preserving valuable base physical information, such as RNA modification and poly(A) length estimation. However, the rapid advancement of ONT devices has set higher standards for analytical software, resulting in potential challenges of software incompatibility and reduced efficiency.
 
 
 
 We present a novel workflow, called FASTdRNA, to manipulate direct RNA-seq data efficiently. This workflow comprises two modules: a data preprocessing module and a data analysis module. The preprocessing data module, dRNAmain, encompasses basecalling, mapping, and transcript counting, which are essential for subsequent analyses. The data analysis module consists of a range of downstream analyses that facilitate the estimation of poly(A) length, prediction of RNA modifications, and assessment of alternative splicing events across different conditions with duplication. The FASTdRNA workflow is designed for the Snakemake framework and can be efficiently executed locally or in the cloud. Comparative experiments have demonstrated its superior performance compared to previous methods. This innovative workflow enhances the research capabilities of direct RNA-seq data analysis pipelines by optimizing existing processes and expanding the scope of analysis.
 
 
 
 The workflow is freely available at https://github.com/Tomcxf/FASTdRNA under an MIT license. Detailed install and usage guidance can be found in the GitHub repository.
 
 
 
 Supplementary data are available at Bioinformatics Advances online.


FASTdRNA: A Workflow for the Analysis of ONT Direct RNA Sequencing

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Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

FASTdRNA: A Workflow for the Analysis of ONT Direct RNA Sequencing