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Spatial separation of the photosynthetic reactions is a key feature of C4 metabolism. In most C4 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 ZmPEPC1 gene expression. Although this region has been well characterized, to date, only few trans-factors involved in the ZmPEPC1 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 ZmPEPC1 upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other ZmPEPC1 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 cis-elements present in the ZmPEPC1 upstream region and one of these cis-elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that ZmOrphan94 is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating ZmPEPC1 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 ZmPEPC1 gene expression.

Plants are unable to physically escape environmental constraints and have, therefore, evolved a range of molecular and physiological mechanisms to maximize survival in an ever-changing environment. Among these, the post-translational modification of ubiquitination has emerged as an important mechanism to understand and improve the stress response. The ubiquitination of a given protein can change its abundance (through degradation), alter its localization, or even modulate its activity. Hence, ubiquitination increases the plasticity of the plant proteome in response to different environmental cues and can contribute to improve stress tolerance. Although ubiquitination is mediated by different enzymes, in this review, we focus on the importance of E3-ubiquitin ligases, which interact with the target proteins and are, therefore, highly associated with the mechanism specificity. We discuss their involvement in abiotic stress response and place them as putative candidates for ubiquitination-based development of stress-tolerant crops. This review covers recent developments in this field using rice as a reference for crops, highlighting the questions still unanswered.