Sugar and Nitrate Sensing: A Multi-Billion-Year Story

AMPK is a highly conserved master regulator of metabolism, which restores energy balance during metabolic stress both at the cellular and physiological levels. The identification of numerous AMPK targets has helped explain how AMPK restores energy homeostasis. Recent advancements, however, demonstrate that regulation of AMPK is also affected by novel contexts, such as subcellular localization and phosphorylation by non-canonical upstream kinases. Notably, the therapeutic potential of AMPK is widely recognized and heavily pursued for treatment of metabolic diseases such as diabetes, but also obesity, inflammation and cancer. Moreover, the recently solved crystal structure of AMPK has shed light both into how nucleotides activate AMPK but, importantly, also into the sites bound by small molecule activators, thus providing a path for improved drugs.

Ions serve as essential nutrients in higher plants and can also act as signaling molecules. Little is known about how plants sense changes in soil nutrient concentrations. Previous studies showed that T101-phosphorylated CHL1 is a high-affinity nitrate transporter, whereas T101-dephosphorylated CHL1 is a low-affinity transporter. In this study, analysis of an uptake- and sensing-decoupled mutant showed that the nitrate transporter CHL1 functions as a nitrate sensor. Primary nitrate responses in CHL1T101D and CHLT101A transgenic plants showed that phosphorylated and dephosphorylated CHL1 lead to a low- and high-level response, respectively. In vitro and in vivo studies showed that, in response to low nitrate concentrations, protein kinase CIPK23 can phosphorylate T101 of CHL1 to maintain a low-level primary response. Thus, CHL1 uses dual-affinity binding and a phosphorylation switch to sense a wide range of nitrate concentrations in the soil, thereby functioning as an ion sensor in higher plants. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

The cistrome is the complete set of transcription factor (TF) binding sites (cis-elements) in an organism, while an epicistrome incorporates tissue-specific DNA chemical modifications and TF-specific chemical sensitivities into these binding profiles. Robust methods to construct comprehensive cistrome and epicistrome maps are critical for elucidating complex transcriptional networks that underlie growth, behavior, and disease. Here, we describe DNA affinity purification sequencing (DAP-seq), a high-throughput TF binding site discovery method that interrogates genomic DNA with in-vitro-expressed TFs. Using DAP-seq, we defined the Arabidopsis cistrome by resolving motifs and peaks for 529 TFs. Because genomic DNA used in DAP-seq retains 5-methylcytosines, we determined that >75% (248/327) of Arabidopsis TFs surveyed were methylation sensitive, a property that strongly impacts the epicistrome landscape. DAP-seq datasets also yielded insight into the biology and binding site architecture of numerous TFs, demonstrating the value of DAP-seq for cost-effective cistromic and epicistromic annotation in any organism.