Calcium-dependent protein kinases in cotton: insights into early plant responses to salt stress

Abstract

Background: Soil salinization is one of the major environmental constraints to plant growth and agricultural production worldwide. Signaling components involving calcium (Ca²⁺) and the downstream calcium-dependent protein kinases (CPKs) play key roles in the perception and transduction of stress signals. However, the study of CPKs in cotton and their functions in response to salt stress remain unexplored.

Results: A total of 98 predicted CPKs were identified from upland cotton (Gossypium hirsutum L. 'TM-1'), and phylogenetic analyses classified them into four groups. Gene family distribution studies have revealed the substantial impacts of the genome duplication events to the total number of GhCPKs. Transcriptome analyses showed a wide distribution of CPKs' expression among different organs. A total of 19 CPKs were selected for their rapid responses to salt stress at the transcriptional level, most of which were also incduced by the thylene-releasing chemical ethephon, suggesting a partal overlap of the salinity and ethylene responses. Silencing of 4 of the 19 CPKs (GhCPK8, GhCPK38, GhCPK54, and GhCPK55) severely compromised the basal cotton resistance to salt stress.

Conclusions: Our genome-wide expression analysis of CPK genes from up-land cotton suggests that CPKs are involved in multiple developmental responses as well as the response to different abiotic stresses. A cluster of the cotton CPKs was shown to participate in the early signaling events in cotton responses to salt stress. Our results provide significant insights on functional analysis of CPKs in cotton, especially in the context of cotton adaptions to salt stress.

Keywords: Calcium dependent protein kinase; Genome-wide identification; Gossypium hirsutum; Transcriptome; Virus-induced gene silencing.

Fig. 1

Phylogenetic analysis of CPK family members in cotton. The phylogenetic tree depicts 98 predicted cotton CPKs and 34 CPKs from A. thaliana, and the un-rooted tree was generated using MEGA6.0 with the neighbor-joining method (1000 bootstrap replicates). Arabidopsis CPKs are denoted with black rhombuses; the numbers represent bootstrap values. All CPKs were classified into one of four groups (groups I-IV); the distinct groups are shown by differently colored branches and backgrounds

Fig. 2

Phylogenetic relationships and exon-intron organization of G. hirsutum CPK genes (GhCPKs). The phylogenetic analysis of G. hirsutum CPK members (left) and the schematic for CPK intron-exon structures (right) of the four different groups. The phylogenetic tree was built using MEGA6.0 with the neighbor-joining method (1000 bootstrap replicates). Gene structures were inferred using GSDS 2.0 and are proportionally displayed according to the scale shown; the black lines indicate introns, while the red boxes represent exons

Fig. 3

Chromosomal distribution of GhCPK genes in the cotton genome. The chromosome numbers are labeled at the top of each chromosome. The red lines on both sides of the chromosomes indicate the coding sequence of the cotton genome and tandem duplications, respectively. GhCPK genes were mapped onto cotton chromosomes and are marked with green lines

Fig. 4

Synteny analysis of G. hirsutum and A. thaliana CPK genes. Chromosomes of G. hirsutum and A. thaliana are shown in a circular form with different colors. Approximate positions of GhCPK and AtCPK genes are labeled with a short gray line on the circle. Synteny between GhCPK and AtCPK genes is shown with colored curves

Fig. 5

Expression patterns of GhCPK genes in 14 representative tissues from G. hirsutum. The heat map, generated with Genesis, shows the hierarchical clustering of GhCPK genes into four groups in vegetative organs (root, stem, and leaf), in floral organs (petal, pistil, and stamen), and at different developmental stages of ovule and fiber tissues. The reads per kilobase of transcript per million mapped reads (RPKM) values were log₁₀ transformed and indicated the expression level of GhCPK genes, while the gradient color (red/black/green) reflects the expression levels (high to low)

Fig. 6

Expression profile of GhCPK genes in response to salt stress. a The cluster analysis was developed using the K-means method on the expression profiles for a total of 98 CPK genes in G. hirsutum. b qPCR analyses of cluster 4 CPK genes expression following NaCl treatment. Cotton seedlings were irrigated with water containing 0 mM, 200 mM or 400 mM NaCl, respectively. Cotton leaves were collected at 0, 1, 3, 6, and 12 h after the treatment. The GhUB7 (ubiquitin 7, Accession: DQ116441) gene was used as an internal control. The error bars indicate the standard deviation estimated from the four replicates

Fig. 7

Abiotic stress and hormonal treatments induced expression of CPK genes. qPCR analysis of the relative expression level of the CPK genes in cluster 4. Cotton seedlings were treated with 100 mM H₂O₂, 100 μM ABA, 1 mM SA, 100 μM MeJA, 200 μM ETH, 5 μM IAA, 0.5 μM GA, 20% PEG, 400 mM NaCl, 4 °C (cold), 37 °C (heat), wounding, or water (as a control), respectively. The GhUB7 gene was used as an internal control. The yellow, green and red bars represent mock, abiotic stresses and hormones treatments, respectively. The green and red arrows emphasize the significantly upregulated expression of target genes under salt and ETH treatments. The error bars indicate the standard deviation estimated from the four replicates (* P < 0.05, ** P < 0.01, t-test)

Fig. 8

Subcellular localization of GhCPKs. Nine GhCPKs (GhCPK8, GhCPK25, GhCPK38, GhCPK54, GhCPK55, GhCPK68, GhCPK88, GhCPK96, and GhCPK97) that were predicted to be involved in salt response were fused with YFP at the C-terminus and transiently expressed in 3-week-old tobacco leaf cells. YFP driven by the CaMV35S promoter was used as a control. Yellow fluorescence was observed 48 h post-Agrobacterium infiltration using confocal microscopy. The plasma membrane labeling by FM4–64 is shown as red fluorescence, the YFP observed in cell nuclei were marked with white arrows (scale bar = 10 μm)

Fig. 9

Functional analysis of cotton CPKs in response to salt stress. a Detection of GhCPK transcripts in TRV:00 and CPK-silenced plants. The cotton UB7 gene was amplified as an internal control. The standard deviations were calculated from the results of three independent experiments (** P < 0.01, t-test). b Leaf disks of control and CPK-silenced plants were incubated in water supplemented with different concentrations of NaCl (0 mM and 400 mM) for 4 days. c Photographs of representative plants of control (TRV:00) and CPK-silenced seedlings (TRV:C8, TRV:C38, TRV:C54, and TRV:C55) under normal condition (Mock) and 400 mM NaCl treatment for 10 days with re-watering. d Measures of plant height and fresh weight, and quantification of Na⁺ and Na⁺/ K⁺ of the aerial portions of TRV:00 and CPK-silenced plants treated with water (control) or 400 mM NaCl for 10 days with re-watering. The standard deviations were calculated from the results of three independent experiments (n ≥ 16, * P < 0.05, ** P < 0.01, t-test)