Genome-wide systematic characterization of the HAK/KUP/KT gene family and its expression profile during plant growth and in response to low-K+ stress in Saccharum

Abstract

Background: Plant genomes contain a large number of HAK/KUP/KT transporters, which play important roles in potassium uptake and translocation, osmotic potential regulation, salt tolerance, root morphogenesis and plant development. Potassium deficiency in the soil of a sugarcane planting area is serious. However, the HAK/KUP/KT gene family remains to be characterized in sugarcane (Saccharum).

Results: In this study, 30 HAK/KUP/KT genes were identified in Saccharum spontaneum. Phylogenetics, duplication events, gene structures and expression patterns were analyzed. Phylogenetic analysis of the HAK/KUP/KT genes from 15 representative plants showed that this gene family is divided into four groups (clades I-IV). Both ancient whole-genome duplication (WGD) and recent gene duplication contributed to the expansion of the HAK/KUP/KT gene family. Nonsynonymous to synonymous substitution ratio (Ka/Ks) analysis showed that purifying selection was the main force driving the evolution of HAK/KUP/KT genes. The divergence time of the HAK/KUP/KT gene family was estimated to range from 134.8 to 233.7 Mya based on Ks analysis, suggesting that it is an ancient gene family in plants. Gene structure analysis showed that the HAK/KUP/KT genes were accompanied by intron gain/loss in the process of evolution. RNA-seq data analysis demonstrated that the HAK/KUP/KT genes from clades II and III were mainly constitutively expressed in various tissues, while most genes from clades I and IV had no or very low expression in the tested tissues at different developmental stages. The expression of SsHAK1 and SsHAK21 was upregulated in response to low-K⁺ stress. Yeast functional complementation analysis revealed that SsHAK1 and SsHAK21 could rescue K⁺ uptake in a yeast mutant.

Conclusions: This study provided insights into the evolutionary history of HAK/KUP/KT genes. HAK7/9/18 were mainly expressed in the upper photosynthetic zone and mature zone of the stem. HAK7/9/18/25 were regulated by sunlight. SsHAK1 and SsHAK21 played important roles in mediating potassium acquisition under limited K⁺ supply. Our results provide valuable information and key candidate genes for further studies on the function of HAK/KUP/KT genes in Saccharum.

Keywords: Evolution; Gene expression; HAK/KUP/KT; Low-K+ stress; Saccharum.

Fig. 1

Nonsynonymous (Ka) and synonymous (Ks) substitution ratios of SsHAKs and their orthologs in sorghum. The Ka/Ks ratio was calculated by the Easy_KaKs calculation program (https://github.com/tangerzhang/FAFUcgb/tree/master/easy_Ka-Ks)

Fig. 2

Phylogeny and schematic diagram for intron/exon organization of HAK/KUP/KT genes from 15 plant species. a Clade II and clade III. b Clade I and clade IV

Fig. 3

Phylogenetic relationships of HAK/KUP/KT families based on the current data for angiosperms

Fig. 4

The expression pattern of HAK/KUP/KT genes based on FPKM in different tissues in different stages in S. officinarum and S. spontaneum

Fig. 5

The expression pattern of HAK/KUP/KT genes based on FPKM across leaf gradients in S. officinarum and S. spontaneum

Fig. 6

The expression pattern of HAK/KUP/KT genes based on FPKM during the diurnal cycles in S. officinarum and S. spontaneum

Fig. 7

a The expression pattern of HAK/KUP/KT genes in Saccharum hybrid YT55 under low-K⁺ stress conditions based on FPKM values. b The relative expression level detected by RT-qPCR

Fig. 8

Phenotypic identification of the yeast mutant strain R5421 transformed with SsHAK1 or SsHAK21

Fig. 9

Schematic models for the roles of HAKs based on gene expression profiles in sugarcane. In the maturing and mature zones of the leaves and stems, HAK7/9/18 were the main expressed genes. Moreover, these genes also presented a diurnal expression pattern. HAK25 was mainly expressed in the maturing and mature zone of leaf tissues, while HAK2 was mainly expressed in the stem. Low-K⁺ stress induced the upregulation of the expression of HAK1 and HAK21. Transcription factors such as DDF2 and JLO may directly bind to the promoters of HAK1/21 to induce gene expression and subsequently promote HAK transporters, such as HAK1 and HAK 21, to acquire K⁺ in roots. HAK1 may be phosphorylated and activated by the CBL1-CIPK23 complex or receptor-like kinase, RUPO (ruptured pollen tube). The K⁺ concentration in the vacuole is highly varied to maintain cellular K⁺ homeostasis. Some HAK transporters, such as HAK10, located in the tonoplast of vacuoles may play a role in regulating the K⁺ concentration in vacuoles