Translatome analyses of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone, is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the inner-most tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR, revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, while BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth. 24 samples of polyribosome-associated mRNA (following 0, 3, and 8 hours of BR treatment, in two biological repetitions), were collected. RNA samples were sequenced on an Illumina HiSeq 2500, to obtain 50 bp single-end reads. These reads were aligned to the TAIR10 assembly of the Arabidopsis thaliana genome, using Tophat2 v2.0.9. Transcript coordinates from the TAIR10 reference set were used to guide the alignment process. After alignment, the numbers of reads per transcript were estimated using HTSeq v0.5.3p3, and DESeq2 v1.2.8 was used to normalize read counts and call differentially expressed genes.

维持生长平衡的机制始终是发育生物学领域的核心研究课题。在植物中，生长平衡依赖于激素信号通路的时空整合，但目前学界对每种激素的确切调控贡献的认知尚处于起步阶段。本研究显示，油菜素类固醇（Brassinosteroid, BR）激素对根分生组织大小的调控效应具有两面性，具体效果取决于其作用的组织位点：当BR分别作用于根分生组织的外层组织——表皮，以及最内层组织——中柱时，可分别延缓和促进干细胞子代细胞的分化起始过程。
为解析这一现象的分子机制，本研究对拟南芥（Arabidopsis thaliana）根系开展了全面的时空翻译组（spatiotemporal translatome）图谱绘制工作。通过对野生型及BR分布模式各异的突变体进行分析，研究团队发现植物存在组织特异性的自主性基因应答反应，这表明BR对生长的调控效应因组织不同而呈现显著差异。BR诱导表达的基因主要在基部分生区的表皮细胞中被检测到，且显著富集于生长素相关基因簇；与之相反，BR抑制表达的基因主要集中在顶端分生区的中柱组织中。此外，研究发现生长素可介导表皮来源的BR信号所产生的促生长效应，而中柱组织中的BR信号则会抵消这一作用。本研究提出，在器官层面，BR的活性与应答反应会因环境背景不同而被反向解读，从而保障植物生长的协调性。
本研究共收集24份多聚核糖体结合mRNA样本：涵盖BR处理0、3、8小时三个时间点，且设置2次生物学重复。使用Illumina HiSeq 2500平台对RNA样本进行测序，获取50 bp单端读段。通过Tophat2 v2.0.9软件将读段比对至拟南芥基因组的TAIR10组装版本，比对过程以TAIR10参考转录本集的转录本坐标作为引导。比对完成后，使用HTSeq v0.5.3p3软件估算每个转录本的读段计数，并通过DESeq2 v1.2.8软件对读段计数进行标准化处理，同时鉴定差异表达基因。