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The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na+) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na+ and K+ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K+ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na+ over K+ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na+ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na+ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na+ channel transport properties.

Nitric oxide (NO), an active signaling molecule in plants, is involved in numerous physiological processes and adaptive responses to environmental stresses. Under high-salt conditions, plants accumulate NO quickly, and reorganize Na + and K + contents. However, the molecular connection between NO and ion homeostasis is largely unknown. Here, we report that NO lowers K + channel AKT1-mediated plant K + uptake by modulating vitamin B6 biosynthesis. In a screen for Arabidopsis NO-hypersensitive mutants, we isolated sno1 (sensitive to nitric oxide 1), which is allelic to the previously noted mutant sos4 (salt overly sensitive 4) that has impaired Na + and K + contents and overproduces pyridoxal 5′-phosphate (PLP), an active form of vitamin B6. We showed that NO increased PLP and decreased K + levels in plant. NO induced SNO1 gene expression and enzyme activity, indicating that NO-triggered PLP accumulation mainly occurs through SNO1-mediated vitamin B6 salvage biosynthetic pathway. Furthermore, we demonstrated that PLP significantly repressed the activity of K + channel AKT1 in the Xenopus oocyte system and Arabidopsis root protoplasts. Together, our results suggest that NO decreases K + absorption by promoting the synthesis of vitamin B6 PLP, which further represses the activity of K + channel AKT1 in Arabidopsis. These findings reveal a previously unidentified pivotal role of NO in modulating the homeostasis of vitamin B6 and potassium nutrition in plants, and shed light on the mechanism of NO in plant acclimation to environmental changes.genetic approach | electrophysiological studies | potassium nutrition N itric oxide (NO) acts as a crucial signaling molecule in various physiological processes in plants, such as seed germination and dormancy (1, 2), root development (3), leaf senescence (4, 5), floral transition (6), stomatal movement (7, 8), iron homeostasis (9, 10), and hormone responses (11,12). NO production is altered when plants are subjected to abiotic or biotic stresses (13,14). High salt, a major environmental factor that limits agriculture yield, induces a quick endogenous NO accumulation in plants (15,16), and triggers enhanced Na + influx and reduced K + absorption in the root (17). Both endogenously produced NO and exogenously applied NO have been proposed to enhance plant salt tolerance (18-21) by attenuating high saltinduced increases in the Na + to K + ratio. Genetic analysis showed that K + nutrition, but not Na + , plays critical role in plant salt tolerance (22). However, the molecular basis of NO effect on K + or Na + content is elusive.K + levels in plant tissues are determined by K + uptake and translocation, which are mediated by a large number of transporters and channels. In Arabidopsis, the K + channel AKT1 (23, 24) and K + transporter AtHAK5 (25) are the two major molecular entities responsible for K + absorption from the environment (26-28). AKT1 contributes to K + acquisition over a wide range of external K + concentrations (10 μM-10 mM), whereas AtHAK5 mediates limited uptake capac...

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.

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