Transcriptome Analysis Unravels Key Factors Involved in Response to Potassium Deficiency and Feedback Regulation of K+ Uptake in Cotton Roots

Abstract

To properly understand cotton responses to potassium (K⁺) deficiency and how its shoot feedback regulates K⁺ uptake and root growth, we analyzed the changes in root transcriptome induced by low K⁺ (0.03 mM K⁺, lasting three days) in self-grafts of a K⁺ inefficient cotton variety (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts with a K⁺ efficient variety (SCRC22/CCRI41). Compared with CCRI41/CCRI41, the SCRC22 scion enhanced the K⁺ uptake and root growth of CCRI41 rootstock. A total of 1968 and 2539 differently expressed genes (DEGs) were identified in the roots of CCRI41/CCRI41 and SCRC22/CCRI41 in response to K⁺ deficiency, respectively. The overlapped and similarly (both up- or both down-) regulated DEGs in the two grafts were considered the basic response to K⁺ deficiency in cotton roots, whereas the DEGs only found in SCRC22/CCRI41 (1954) and those oppositely (one up- and the other down-) regulated in the two grafts might be the key factors involved in the feedback regulation of K⁺ uptake and root growth. The expression level of four putative K⁺ transporter genes (three GhHAK5s and one GhKUP3) increased in both grafts under low K⁺, which could enable plants to cope with K⁺ deficiency. In addition, two ethylene response factors (ERFs), GhERF15 and GhESE3, both down-regulated in the roots of CCRI41/CCRI41 and SCRC22/CCRI41, may negatively regulate K⁺ uptake in cotton roots due to higher net K⁺ uptake rate in their virus-induced gene silencing (VIGS) plants. In terms of feedback regulation of K⁺ uptake and root growth, several up-regulated DEGs related to Ca²⁺ binding and CIPK (CBL-interacting protein kinases), one up-regulated GhKUP3 and several up-regulated GhNRT2.1s probably play important roles. In conclusion, these results provide a deeper insight into the molecular mechanisms involved in basic response to low K⁺ stress in cotton roots and feedback regulation of K⁺ uptake, and present several low K⁺ tolerance-associated genes that need to be further identified and characterized.

Keywords: cotton; grafting; nutrient transporter; potassium deficiency; transcription factor.

Figure 1

Effects of potassium (K⁺) deficiency on CCRI41 (a K⁺ inefficient cotton variety) self-grafts (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts (SCRC22/CCRI41) with SCRC22 (a K⁺ efficient variety). Grafting was performed hypocotyl-to-hypocotyl (a) when the cotyledons of rootstock just fully expanded. Grafts were subjected to low K⁺ (LK, 0.03 mM K⁺) at the three-leaf stage, with 2.5 mM K⁺ as control (CK). After 16 days, all leaves were photographed (b), and the chlorophyll content in the third (3rd L), fourth (4th L) and fifth (5th L) leaves was measured (c). The dry weight of roots, stem and leaves was recorded (d), and the root-shoot ratio was calculated (e). K⁺ concentration (f) and K⁺ accumulation (g) in roots, stem and leaves were determined or calculated. CK: Control; LK: Low K⁺ treatment. The data are shown as means ± SD of three replicates (n = 3); * and ** indicate significant differences at 5% and 1% level, respectively.

Figure 2

Transcriptome relationships among the roots of CCRI41 self-grafts (CCRI41/CCRI41, scion/rootstock) and reciprocal grafts (SCRC22/CCRI41) under potassium (K⁺) deficiency. (a) Cluster dendrogram of the root transcriptomes. (b) Number of differentially expressed genes (DEGs) in the roots of CCRI41/CCRI41 and SCRC22/CCRI41. (c) Venn diagram showing shared and unique DEGs in both grafts. CR and LR: Roots from CK and LK-treated plants; 41/41: CCRI41/CCRI41; 22/41: SCRC22/CCRI41; 1, 2, and 3: Number of replicates. R common: Common DEGs in the roots of both grafts. R22/41 unique: The DEGs specifically identified in the roots of SCRC22/CCRI41.

Figure 3

The differentially expressed genes (DEGs) involved in Ca²⁺ (calcium) signaling in R common (a) and R_22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K⁺) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41.

Figure 4

The differently expressed genes (DEGs) involved in ROS (reactive oxygen species) signaling in R common (a) and R_22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K⁺) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41.

Figure 5

The differentially expressed genes (DEGs) involved in phytohormone signaling in R common (a) and R_22/41 unique (b) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K⁺) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41. Red and purple boxes contain genes that were up- and down-regulated, respectively. The red and blue colored boxes contain genes that were oppositely regulated in both grafts, the left and right boxes were assigned to CCRI41/CCRI41 and SCRC22/CCRI41, respectively. The “DNA” in diagram represents undefined genes.

Figure 6

The differentially expressed genes (DEGs) related to transporters in R common (a) and R_22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K⁺) deficiency. R22/41 unique: The DEGs specifically identified in roots of SCRC22/CCRI41.

Figure 7

The predicted transcription factors (TFs) that have binding sites in the 2-kb promotor region of the K⁺ transporter gene GhHAK5 (Gh_D01G1760) in R common (a) and R_22/41 unique (b) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K⁺) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41.

Figure 8

Two members of ethylene response factor (ERF) TFs, GhERF15 and GhESE3, negatively regulate potassium (K⁺) uptake in cotton roots. GhERF15 and GhESE3 were silenced in the variety SCRC22 using agrobacterium-mediated virus-induced gene silencing (VIGS) at the cotyledonary stage. The relative expression of GhERF15 (a) and GhESE3 (b) indicates that they were silenced in both A and D subgenome. The seedlings at three-leaf stage were moved into K⁺-starvation solutions for 48 h, then transferred to measuring solution with 0.08 mM K⁺ to determine the net K⁺ uptake rate (c,d). * and ** indicate significant differences at 5% and 1% level, respectively.

Figure 9

A model of transcription regulation involved in response to potassium (K⁺) deficiency and feedback regulation of K⁺ uptake and root growth in cotton. Genes within the light grey box show the same responses to low K⁺ stress in CCRI41 (a K⁺ inefficient cotton variety) self-grafts (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts (SCRC22/CCRI41) with SCRC22 (a K⁺ efficient variety); while genes within the white box represent the components involved in feedback regulation of K⁺ uptake and root growth under K⁺ deficiency, they were specifically identified in the roots of SCRC22/CCRI41 or oppositely regulated by low K⁺ in CCRI41/CCRI41 and SCRC22/CCRI41. The red and blue highlighted genes were up- and down-regulated by low K⁺ respectively.