The Potassium Utilization Gene Network in Brassica napus and Functional Validation of BnaZSHAK5.2 Gene in Response to Potassium Deficiency

Abstract

Potassium, an essential inorganic cation, is crucial for the growth of oil crops like Brassica napus L. Given the scarcity of potassium in soil, enhancing rapeseed's potassium utilization efficiency is of significant importance. This study identified 376 potassium utilization genes in the genome of B. napus ZS11 through homologous retrieval, encompassing 7 functional and 12 regulatory gene families. These genes are unevenly distributed across 19 chromosomes, and the proteins encoded by these genes are mainly localized in the cell membrane, vacuoles, and nucleus. Microsynteny analysis highlighted the role of small-scale replication events and allopolyploidization in the expansion of potassium utilization genes, identifying 77 distinct types of cis-acting elements within their promoter regions. The regulatory mechanisms of potassium utilization genes were provided by analyses of transcription factors, miRNA, and protein interaction networks. Under low potassium stress, the potassium utilization genes, particularly those belonging to the KUP and CBL families, demonstrate pronounced co-expression. RNA-seq and RT-qPCR analysis identified the BnaZSHAK5.2 gene, which is a high-affinity potassium ion transporter, playing a crucial role in the stress response to potassium deficiency in B. napus, as its expression is strongly induced by low potassium stress. A functional complementation study demonstrates that the BnaZSHAK5.2 gene could rescue the primary root growth of the Athak5 mutant under low potassium conditions, confirming its role in response to low potassium stress by sustaining root development.

Keywords: BnaZSHAK5.2; Brassica napus L.; gene function; potassium utilization gene network.

Figure 1

Subcellular localization of K+ utilization-related proteins in B. napus.

Figure 2

Analysis of gene evolution of K+ utilization-related genes in B. napus. (A): Chromosome mapping of K+ utilization genes. (B): Number of K+ utilization genes on each chromosome. (C): Gene duplication event, upper right corner shows duplication events of all K+ utilization genes, and bar chart shows duplication events of functional and regulatory genes of all K+ utilization genes. (D): Linear relationship analysis of B. napus K+ utilization pathway genes and homologous genes in B. rapa and B. oleracea genomes.

Figure 3

Cis-acting elements of K+ utilization genes in B. napus.

Figure 4

K+ deficiency stress expression profile of K+ utilization genes in B. napus. LK: K+ deficiency treatment. “1 d”, “3 d”, “5 d”, “7 d”, and “12 d” represent number of days after K+ deficiency treatment. L: leaf; R: root.

Figure 5

Co-expression network of K+ utilization genes in B. napus across different development stages. Orange circles represent transcription factors, and blue “V” shape graphics represent structural genes.

Figure 6

Gene expression pattern under potassium deficiency. (A): Differential expression of BnaZSKUPs under K+ deficiency stress. (B): Expression changes of BnaZSHAKs in potassium deficiency. * indicates that the p value is less than 0.5 and ** is less than 0.01.

Figure 7

Phenotype analysis of root hair of transgenic Arabidopsis. (A): Phenotype of Arabidopsis root length. (B): Statistics of Arabidopsis root length. a: athak5 mutant; b: WT; c: BnaZSHAK5.2p::BnaZSHAK5.2; LK: K+ deficiency; 10 μm and 50 μm represent concentration of K+ in medium; CK: full nutrition. ** indicates that the p value is less than 0.01.