Integrated transcriptomic and metabolomic analyses reveal key metabolic pathways in response to potassium deficiency in coconut (Cocos nucifera L.) seedlings

Abstract

Potassium ions (K⁺) are important for plant growth and crop yield. However, the effects of K⁺ deficiency on the biomass of coconut seedlings and the mechanism by which K⁺ deficiency regulates plant growth remain largely unknown. Therefore, in this study, we compared the physiological, transcriptome, and metabolite profiles of coconut seedling leaves under K⁺-deficient and K⁺-sufficient conditions using pot hydroponic experiments, RNA-sequencing, and metabolomics technologies. K⁺ deficiency stress significantly reduced the plant height, biomass, and soil and plant analyzer development value, as well as K content, soluble protein, crude fat, and soluble sugar contents of coconut seedlings. Under K⁺ deficiency, the leaf malondialdehyde content of coconut seedlings were significantly increased, whereas the proline (Pro) content was significantly reduced. Superoxide dismutase, peroxidase, and catalase activities were significantly reduced. The contents of endogenous hormones such as auxin, gibberellin, and zeatin were significantly decreased, whereas abscisic acid content was significantly increased. RNA-sequencing revealed that compared to the control, there were 1003 differentially expressed genes (DEGs) in the leaves of coconut seedlings under K⁺ deficiency. Gene Ontology analysis revealed that these DEGs were mainly related to "integral component of membrane," "plasma membrane," "nucleus", "transcription factor activity," "sequence-specific DNA binding," and "protein kinase activity." Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the DEGs were mainly involved in "MAPK signaling pathway-plant," "plant hormone signal transduction," "starch and sucrose metabolism," "plant-pathogen interaction," "ABC transporters," and "glycerophospholipid metabolism." Metabolomic analysis showed that metabolites related to fatty acids, lipidol, amines, organic acids, amino acids, and flavonoids were generally down-regulated in coconut seedlings under K⁺ deficiency, whereas metabolites related to phenolic acids, nucleic acids, sugars, and alkaloids were mostly up-regulated. Therefore, coconut seedlings respond to K⁺ deficiency stress by regulating signal transduction pathways, primary and secondary metabolism, and plant-pathogen interaction. These results confirm the importance of K⁺ for coconut production, and provide a more in-depth understanding of the response of coconut seedlings to K⁺ deficiency and a basis for improving K⁺ utilization efficiency in coconut trees.

Keywords: Cocos nucifera L.; metabolome; physiology; potassium deficiency; transcriptome (RNA-seq).

Figure 1

Effects of K⁺ deficiency on coconut seedlings. (A) Phenotype of coconut seedlings after 30 days of K⁺ treatment. (B) Changes in SPAD, SP, SS, and CF contents under K⁺ deficiency for 30 consecutive days. *P ≤ 0.05, **P ≤ 0.01. Bars represent SDs.

Figure 2

MDA, Pro, IAA, GA, ABA, and ZR contents and SOD, CAT, and POD activities in K₀ vs. K_ck. *P ≤ 0.05, **P ≤ 0.01. Bars represent SDs.

Figure 3

Top 20 GO pathways enriched in DEGs in K₀ vs. K_ck, including the three categories of BP, CC, and MF. (A) BP terms enriched in up-regulated DEGs. (B) CC terms enriched in up-regulated DEGs. (C) MF enriched in up-regulated DEGs. (D) BP terms enriched in down-regulated DEGs. (E) CC terms enriched in down-regulated DEGs. (F) MF terms enriched in down-regulated DEGs. The X-axis (GeneNum) represents the number of genes of interest annotated in the entry, the Y-axis indicates each term entry. The color of the column represents the P -value of the hypergeometric test.

Figure 4

KEGG pathway enrichment of DEGs in K₀ vs. K_ck. (A) Top 20 KEGG pathways enriched in DEGs in K₀ vs. K_ck. (B) Top 20 KEGG pathways enriched in up-regulated DEGs in K₀ vs. K_ck. (C) Top 20 KEGG pathways enriched in down-regulated DEGs in K₀ vs. K_ck. The X-axis (GeneNum) represents the number of genes of interest annotated in the entry, the Y-axis indicates each pathway entry. The color of the column represents the P-value of the hypergeometric test. (D) Top 20 KEGG pathway enrichment bubble chart of all DEGs in K₀ vs. K_ck. (E) Bubble chart of the top 20 KEGG pathways enriched up-regulated DEGs in K₀ vs. K_ck. (F) Bubble chart of the top 20 KEGG pathways enriched in down-regulated DEGs in K₀ vs. K_ck. The X-axis (GeneRatio) represents the proportion of the gene of interest annotated in the entry to the number of all DEGs, the Y-axis indicates each pathway entry. Dot size represents the number of DEGs annotated in the pathway, and dot color represents the P-value of the hypergeometric test. (G) Scatter plot of top 20 KEGG pathway enrichment of all DEGs in K₀ vs. K_ck. Each circle represents a KEGG pathway, the Y-axis indicates the name of the pathway, and the X-axis represents the enrichment factor, which is the ratio of DEGs annotated to a certain pathway to total genes annotated to the pathway. The color of the circle represents the q-value, which is the P-value after multiple hypothesis test correction.

Figure 5

KEGG pathway enrichment network of DEGs in the top five enriched pathways in K₀ vs. K_ck. (A) up-regulated DEGs, (B) down-regulated DEGs, and (C) all DEGs. Line colors represent different pathways, and gene node colors represent multiple differences.

Figure 6

DEGs related to the MAPK signaling pathway - plant, starch and sucrose metabolism, plant hormone signal transduction, plant-pathogen interaction, glycerophospholipid metabolism, alpha-linolenic acid metabolism, circadian rhythm - plant. Changes in relative expression under K⁺ deficiency (log2FC based on mean RPKM in K₀ vs. K_ck) are shown as a color gradient from low (green) to high (red). Log2(FC) > 0 indicates upregulation, log2(FC) < 0 indicates downregulation, and log2(FC) = 0 indicates unchanged expression.

Figure 7

Analysis of DAMs in K₀ vs. K_ck. (A) Volcano plat of the DAMs. Each dot represents a metabolite. The X-axis represents the change in each substance (Log2), the Y-axis represents the P-value of the t-test (Log10), and the scatter size represents the variable importance of projection VIPvalue of the OPLS-DA model. down-regulated DAMs are indicated in blue, up-regulated DAMs are shown in red, and metabolites with insignificant differences are shown in grey. The first five qualitative metabolites were selected and labeled in the figure, based on their P-value. (B) Three-dimensional diagram of PCA. (C) OPLS-DA score model chart. R2X and R2Y represent the interpretation rate of the built model to the X and Y matrices, respectively, wherein the X matrix is the model input, i.e., the metabolite quantitative matrix, the Y matrix is the model output, i.e., the sample grouping matrix, and Q2 represents the prediction ability of the model, i.e., whether the built model can distinguish the correct sample grouping based on metabolite expression. The closer R2Y and Q2 are to 1 in the index, the more stable and reliable the model is. This model can be used to screen DAMs. Generally, Q2 > 0.5 indicates an effective model, and Q2 > 0.9 reflects an excellent model. (D) OPLS-DA model validation chart. The X-axis represents the similarity with the original model, the Y-axis represents the value of R2Y or Q2 (where R2Y and Q2 taken as 1 in the abscissa are the values of the original model), the blue and red points represent R2Y and Q2 of the model after Y replacement, respectively, and the dotted line is the fitted regression line. (E) Clustering heat map of the 164 DAMs. (F) Histogram of classification changes of different metabolites.

Figure 8

Metabolite pathway analysis in K₀ vs. K_ck. (A) Classification diagram of different metabolite pathways in each group. The X-axis indicates the number of DAMs annotated to a pathway, and the Y-axis indicates the name of the pathway. (B) Enrichment map of DAMs in KEGG pathways. The X-axis represents the ratio of the DAMs in a pathway to all DAMs with pathway annotation. (C) KEGG enrichment network diagram of DAMs. The light-yellow node indicates the pathway, and the small node connected to it indicates the specific metabolite annotated to the pathway. Color depth represents Log2(FC). The figure shows up to five pathways.

Figure 9

Analysis of KEGG pathways significantly enriched in DEGs/DAMs in K₀ vs. K_ck. (A) Bubble diagram of the top 30 KEGG pathways significantly enriched in DEGs/DAMs. The X-axis represents the Gene Enrich factor, the diff/background of genes in this pathway, and the Y-axis indicates the enrichment pathway. Dot size represents the significance of enrichment of the annotated DAMs in the pathway; the larger the dot, the more enriched it is. Dot color represents the significance of enrichment of DEGs in this pathway; the deeper the color, the more enriched they are. (B) Histogram of the top 30 KEGG pathways with significant enrichment of DEGs/DAMs. Each column represents a KEGG pathway. Yellow represents the transcriptome and blue represents the metabolome. Pathway names are indicated on the Y-axis, and the X- axis indicates the significance of enrichment of each pathway, that is, the FDR value taking the logarithm of the FDR.

Figure 10

DEG and DAM correlation network diagram of 22 KEGG pathways in K₀ vs. K_ck. Circles represent metabolites, boxes represent genes, and values on the line represent correlation coefficients. Positive and negative correlation is indicated in red and green, respectively. The larger the correlation coefficient, the wider the line and the darker the color. The number in each ellipse indicates the ID of the KEGG pathway corresponding to the network diagram.

Figure 11

Profiles of DEGs and DAMs in flavonoid biosynthetic pathways in K₀ vs. K_ck. The boxes in the pathway represent DEGs or DAMs. Red and green represent up- and down-regulated genes, respectively. Yellow and blue represent up- and down-regulated metabolites, respectively. Boxes of the same color represent the relevant pathway.