Effect of low-K + stress on the phenotype, K+ content, and relative expression of marker genes in sweet potato seedlings
To identify the phenotype of sweet potato seedlings under K+-deficiency condition. The low-K+ stress (LK, 0 mM K+) was applied at two-leaf stage. After 14 days, the K+-deficient phenotypes were evaluated. Compared with control seedlings (HK, 1 mM K+), sweet potato seedlings were remarkably inhibited in growth and exhibited chlorosis, which was first observed in older leaves under K+-deficiency condition (Figure 1a). The typical potassium deficiency phenotype was consistent with that of other crops [45, 46], and the K+ content was significantly lower under low-K+ stress (Figure 1b). Moreover, we determined the relative expression of the low-K+ stress marker genes to further support our phenotype. The CIPK23 and HAK5 genes were reported to respond to low-K+ stress, and play crucial roles particularly under K+-deficient conditions [15, 47]. Our results showed that two important genes were noticeable upregulated, especially in sweet potato roots under K+ deficiency (Figure 1c and 1d). The phenotype and K+ content analyses showed that the sweet potato seedlings suffered from low-K stress (Figure 1a and 1b). Therefore, these results proved that the material is valid for obtaing transcriptome profiles of sweet potato under K+-deficient conditions.
Transcriptome profiling in response to K+ deficiency
To assess gene expression in sweet potato roots under K+-deficient condition, we conducted the high-throughput transcriptome sequence analysis without a genome [44]. To ensure reliability of the transcriptome data, three biological replicates were analyzed for each treatment. The roots of seedlings of Taizhong6 which is the variety cultivated in china were treated under sufficient K+ and low-K+ stress for 14 days and RNA was extracted to obtain transcriptomic profiles. The quality of the sequencing data was sufficient to support further transcriptome analysis by paired-end sequencing using an Illumina NovaSeq6000 sequencing platform. After quality control, aapproximately 22,350,759 and 26,153,929 clean reads were respectivly obtained und low K+ and high K+ condition (Table S1).
Trinity-v2.4.0 software was used to assemble clean reads and obtain unigenes. After analyzing the open-reading frame (ORF) findings, singel-nucleotide polymorphisms (SNP), and simple sequence repeats (SSR), the genome was mapped for annotation and expression analysis. The quality of the assembly is closely related to the length and number of unigenes. In our study, the unique gene length distributed through a wide range and those over 3,000 bp accounted for about 3.06% (Figure S1a). The unigenes were annotated in multiple databases, such as, NR database (the NCBI non-redundant protein sequences); Swissprot(a manually annotated and non-redundant protein sequence database); KEGG (Kyoto Encyclopedia of Genes and Genomes); KOG (Clusters of Orthologous Groups of proteins). The venn diagram shows the annotated genes for each database (Figure S1b).
To investigate differences among the transcriptome sequence data, the hierarchical cluster analysis of different expression genes during K+ sufficient and K+ deficient conditions was conducted (Figure 2a). Statistical results showed that approximately 336 genes were observably upregulated and approximately 223 were downregulated in the low-K+ treatment (Figure 2b). The numbers and fold changes of different expression genes under low K+ are clearly displayed in the MA plot, and the red and green dots indicate obviously differentially expressed genes (Figure 2c).
Transcriptome sequencing identified DEGs in sweet potato seedlings under low-K + stress
In this study, 559 DEGs were observed under low-K+ stress and we choosed some DEGs showed in Table 1. Including transports, transcription factors, cell wall related genes, disease resistance related genes, kinase, E3 ubiquitin ligase and uclacyanin. Some of these genes are closely related with K+. For example, K+ and NO3- are reported to cooperative uptake. In our study, high affinity nitrate transporter 2.4 was upregulated, which implys the nitrate transporter may be involved in K+ homeostasis. To validate the quality of the gene activity profiles, eight genes were randomly selected to compare their Fragments per kilobase of exon per million fragments mapped (FPKM ) reads and qPCR data. Four upregulated and four downregulated genes were randomly selected from different expression genes (DEGs) to conduct qPCR experiment (Table 2). The functional annotation showed that the eight DEGs may function in different pathways to respond to low-K+ stress. These unigenes are involved in sugar metabolism pathways, plant cell wall activity, cell division, pathogenesis-related activity, and cytochrome processes (Table 1). Real-time PCR analyses further confirmed that the expression of the selected genes and the trend of gene expression changes determined by the two different approaches were largely consistent (Figure 3). Thus, the DEGs determined in this study can be considered highly accurate.
Gene ontology analysis of DEGs in sweet potato seedlings in response to K + deficiency
To evaluate the potential functions of these DEGs in response to K+ deficiency, gene ontology (GO) analysis was performed by mapping each DEG into the records of the GO database with an adjusted p-value < 0.05 as significant enrichment. We chose the top 20 GO terms for further analysis of biological processes, cellular component and molecular function processes (Figure 4). In the GO classification of biological, the cellular catabolic processes and oxidation-reduction process were the significantly riched group, which indicated that the Taizhong6 variety had a wide range of catabolic and oxidation-reduction activities under low-K+ stress. Within the “cellular component” calssification, the intracellular part, cytoplasm, intracellular organelle, and organelle part were prominently represented. Within the “molecular function” classification, the main functional group of the DEGs were genes related to metal ion binding, protein binding, DNA binding, and nucleic acid binding of TFs. Most of these processes were closely related to a response to K+-deficiency.
KEGG analysis of DEGs in sweet potato seedlings in response to K + deficiency
To identify the pathways in which the DEGs are likely to be involved, we performed the pathway analysis with the KEGG (https://www.kegg.jp/ ) database. The top 20 pathways are shown in Figure 5. The analysis showed that the retinol metabolism pathway, tyrosine metabolism, metabolism of xenobiotics by cytochrome P450, steroid hormone biosynthesis, and other glycan degradations were significantly enriched (Table S3). Previous studies report that low-K+ stress affects the color of fruits and the quality of crops [28, 31, 48]. The cultivar Taizhong6 used in this study is a carotenoid-rich sweet potato variety; we found that some important DEGs were observably involved in cytochrome or retinol metabolism pathways under K+ deficiency condition (Figure 5, Table 2). This finding suggests that low-K+ stress is closely related to the production of carotenoids, and these annotations provide a valuable resource for investigating the response mechanism of sweet potato under low K+.