Regulation Of K⁺/NO₃^-Translocation By Transcription Factor MYB59 Under Low K⁺ Stress In Arabidopsis

Potassium(K)and nitrogen(N)are essential macronutrients for plant growth and development by involving numerous regulatory and metabolic processes in plant life cycle.K+ functions in enzyme activation,osmotic regulation,and electrical neutralization,and nitrogen is part of carbon compounds and constitutes amino acids,proteins,nucleic acids,etc.It is known that absorption and transport of K+ and NO3-are somehow closely related,but coordinating mechanism between K+ and NO3-remains unclear.Our previous work has revealed that the nitrate transporter NRT1.5 also functions as a K+ transporter and plays roles in regulating the coordinated transport of K+ and NO3-from root to shoot in Arabidopsis.It is also known that NRT1.5 is regulated at the transcriptional level in response to external K+ and/or NO3-concentration changes.Following low K+ or low NO3-treatments,the transcription of NRT1.5 is down-regulated and the root-to-shoot KT/NO3-co-transport is inhibited.In this dissertation work,a MYB transcription factor MYB59 is identified as a transcriptional regulator of NRT1.5 in Arabidopsis response to external K+availability.An Arabidopsis low-K+-sensitive mutant lks3 was isolated and its shoots became yellow earlier than wild-type plants under low K+ stress.Map-based cloning and phenotype analyses showed that LKS3 encodes a transcriptional factor MYB59.The myb59 mutant showed low-K+-sensitive phenotype similar to lks3 and its complementation lines rescued the phenotype of myb59 mutant.These data suggested that the transcription factor MYB59 is involved in Arabidopsis responses to low K+ stress.MYB59 transcription undergoes conserved alternative splicing in Arabidopsis and only the longest transcript(MYB59.3)could restore the phenotype of myb59 to wild type,suggesting that MYB59.3 is the functional transcript in plants.Ion content measurement indicated that the roots of myb59 mutants accumulated more K+ and NO3-ions compared with wild-type plants under LK conditions.In contrast,the K+ and NO3-contents in mutant shoots were significantly reduced.These results suggest that MYB59 is involved in regulation of root-to-shoot K+/NO3-co-translocation under LK conditions.RNA-seq and RT-qPCR results showed that the transcription of NRT1.5 was significantly reduced in myb59 mutant plants,suggesting that MYB59 positively regulate NRT1.5 transcription.The nrt1.5 mutants displayed the very similar sensitive phenotype as myb59 mutants on LK medium,and root-to-shoot transport of K+/NO3-is impaired in both myb59 and nrtl.5 mutants.The myb59 nrt1.5 double mutant had the similar sensitive phenotype and ion contents as their single mutants,indicating that MYB59 and NRT1.5 should be in the same regulatory pathway.In addition,the low-K+-sensitive phenotype of either nrt1.5 or myb59 mutant could be complemented by transgenically expressing pPHO1:NRT1.5 in either mutant,demonstrating NRT1.5 as the down-stream target gene of MYB59.The NRT1.5 promoter region contains some cis-elements that may represent binding sites of MYB59.ChIP and EMSA results showed that MYB59.3 can directly bind to the NRT1.5 promoter either in vivo or in vitro.RT-qPCR results showed that low K+ stress repressed MYB59 and NRT1.5 transcription.And the semi-in vivo protein degradation experiment showed that MYB59.3 was degraded rapidly after LK treatment.In summary,this dissertation work demonstrated that the transcription factor MYB59 positively regulates NRT1.5 expression to coordinate K+/NO3-transport in Arabidopsis under normal K+ supply.When plants are subjected to K+ deficient stress,MYB59 is down-regulated and the transcription of NRT1.5 is subsequently inhibited.As a result,the root-to-shoot K+/NO3-translocation is reduced to balance the K+/NO3-distribution between root and shoot.