Górska Alicja M, Gouveia Paulo, Borba Ana Rita, Zimmermann Anna, Serra Tânia S, Carvalho Pedro, Lourenço Tiago F, Oliveira M Margarida, Peterhänsel Christoph, Saibo Nelson J M
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
Front Plant Sci. 2021 Apr 8;12:559967. doi: 10.3389/fpls.2021.559967. eCollection 2021.
Spatial separation of the photosynthetic reactions is a key feature of C metabolism. In most C plants, this separation requires compartmentation of photosynthetic enzymes between mesophyll (M) and bundle sheath (BS) cells. The upstream region of the gene encoding the maize PHOSPHOENOLPYRUVATE CARBOXYLASE 1 (ZmPEPC1) has been shown sufficient to drive M-specific gene expression. Although this region has been well characterized, to date, only few -factors involved in the gene regulation were identified. Here, using a yeast one-hybrid approach, we have identified three novel maize transcription factors ZmHB87, ZmCPP8, and ZmOrphan94 as binding to the upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other regulators previously reported, ZmbHLH80 and ZmbHLH90. Trans-activation assays in maize M protoplasts unveiled that ZmHB87 does not have a clear transcriptional activity, whereas ZmCPP8 and ZmOrphan94 act as activator and repressor, respectively. Moreover, we observed that ZmOrphan94 reduces the trans-activation activity of both activators ZmCPP8 and ZmbHLH90. Using the electromobility shift assay, we showed that ZmOrphan94 binds to several -elements present in the upstream region and one of these -elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating transcript level in the BS cells. Based on both this and our previous work, we propose a model underpinning the importance of a regulatory mechanism within BS cells that contributes to the M-specific gene expression.
光合作用反应的空间分离是C代谢的一个关键特征。在大多数C4植物中，这种分离需要光合酶在叶肉（M）细胞和维管束鞘（BS）细胞之间进行区室化。已证明编码玉米磷酸烯醇式丙酮酸羧化酶1（ZmPEPC1）的基因上游区域足以驱动叶肉特异性基因表达。尽管该区域已得到充分表征，但迄今为止，仅鉴定出少数参与该基因调控的因子。在此，我们使用酵母单杂交方法，鉴定出三种新的玉米转录因子ZmHB87、ZmCPP8和ZmOrphan94与该上游区域结合。玉米M原生质体中的双分子荧光互补分析表明，ZmOrphan94形成同二聚体，并与ZmCPP8以及先前报道的另外两个调节因子ZmbHLH80和ZmbHLH90相互作用。玉米M原生质体中的反式激活分析表明，ZmHB87没有明显的转录活性，而ZmCPP8和ZmOrphan94分别作为激活剂和抑制剂起作用。此外，我们观察到ZmOrphan94降低了激活剂ZmCPP8和ZmbHLH90的反式激活活性。使用电泳迁移率变动分析，我们表明ZmOrphan94与上游区域中存在的几个元件结合，并且这些元件之一与ZmbHLH90结合位点重叠。基因表达分析表明，ZmPEPC1在维管束鞘细胞中优先表达，这表明ZmOrphan94是下调维管束鞘细胞中ZmPEPC1转录水平的转录调控网络的一部分。基于此项研究以及我们之前的工作，我们提出了一个模型，该模型强调了维管束鞘细胞内一种调控机制对于叶肉特异性ZmPEPC1基因表达所起的重要作用。
