Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

Abstract

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na⁺/H⁺ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

Keywords: CBL-interacting protein kinase; abiotic stress; calcineurin B-like protein; cassava; signal pathway.

Figure 1

Multiple sequence alignment between cassava and Arabidopsis CBL proteins. Sequence alignment was performed using DNAMAN 5.0 software. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. The four EF hand motifs are indicated by black boxes. The myristoylation sites are in the red box.

Figure 2

Multiple sequence alignment between cassava and Arabidopsis CIPK proteins. Sequence alignment was performed using DNAMAN 5.0 software. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. The NAF/FISL and PPI motifs are indicated by black boxes. The conserved amino acids in the motifs are indicated by asterisk.

Figure 3

Conserved motifs (A) and gene structures (B) of cassava CBL proteins and genes, respectively. (A) The conserved motifs were identified using the MEME program and are arranged according to the phylogenetic tree. Different motifs are highlighted with different color boxes. The length of boxes corresponds to motif length. (B) The gene structures were drawn using the GSDS program and arranged according to the phylogenetic tree. The yellow boxes represent exons, the blue boxes represent upstream and downstream UTRs and the lines represent introns.

Figure 4

Conserved motifs (A) and gene structures (B) of cassava CIPK genes. (A) The conserved motifs were using the MEME program and arranged corresponding to the phylogenetic tree. Different motifs are highlighted with different color boxes. The length of boxes corresponded to motif length. (B) The gene structures were drawn using the GSDS program and arranged corresponding to the phylogenetic tree. The yellow boxes represent exons, the blue boxes represent upstream and downstream UTRs and the lines represent introns.

Figure 5

Expression profile analyses of MeCBL (A) and MeCIPK (B) genes in cassava tissues using the RT-PCR method. “R” represents roots from mature plants or seedlings, “S” represents stems from mature plants or seedlings, “YL” and “OL” represent young leaves and old leaves of mature plants, respectively, “F” represents flowers, “SR” represents storage roots, and “L” represents the seedlings leaves.

Figure 6

Expression analyses of MeCBL genes in roots (A) and leaves (B) responding to abiotic stresses, including salt (200 mM NaCl), drought (20% PEG6000), cold (4°C) and heat (42°C) treatments. Data are the means of three biological replicates ± SE at 3 h and 9 h time points standardized at 0 h.

Figure 7

Expression analyses of MeCIPK genes in roots responding to abiotic stresses, including salt (200 mM NaCl), drought (20% PEG6000), cold (4°C) and heat (42°C) treatments. Data are the means of three biological replicates ± SE at 3 and 9 h time-point standardized at 0 h.

Figure 8

Expression analyses of MeCIPK genes in leaves responding to abiotic stresses, including salt (200 mM NaCl), drought (20% PEG6000), cold (4°C) and heat (42°C) treatments. Data are the means of three biological replicates ± SE at 3 h and 9 h time-point standardized at 0 h.

Figure 9

The effect of salinity stress on yeast growth. Transgenic and untransformed yeast cells were pre-cultured to saturation, and serial 10-fold dilutions of yeast cells were spotted on AP plates without or with NaCl as described. After 5 days, the growth of yeast cells on plates was recorded. MeSOS1, AXT3K strain transformed with the Na⁺/H⁺ antiporter gene MeSOS1; MeSOS1+MeCIPK24, AXT3K strain transformed with MeSOS1 and MeCIPK24 genes; MeSOS1+MeCIPK24+MeCBL10, AXT3K strain transformed with MeSOS1, MeCIPK24 and MeCBL10 genes; AXT3K, untransformed AXT3K strain.

Figure 10

Potential CBL-CIPK signaling networks in cassava response to abiotic stresses. Blue arrows represent the interaction relationships between MeCBL and MeCIPK proteins, as indicated by protein-protein interaction data and red arrows represent observed gene expression responses to abiotic stresses.