The rice OsCCC1 is a member of the cation-Cl- cotransporter (CCC) family and plays a significant role in K+ and Cl－ homeostasis and rice plant development.
- Xiang-Qiang Kong et al. have cloned an Oryza sativa cDNA encoding for a member of the cation–Cl- cotransporter (CCC) family. Plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K+ –Cl- cotransporters than to other cation–Cl- cotransporters. In plants CCCs are involved in the homeostasis of Cl-.
- OsCCC1 was primarily involved in K+ homeostasis and Cl- homeostasis, it might be an K+ -Cl- cotransporter. It is a compact phylogenetic cluster with highly conserved genes that shows the highest similarity to animal KCCs. OsCCC1 plays a significant role in ion homeostasis and rice development under saline conditions. The analysis of OsCCC1 RNAi lines designed by Xiang-Qiang Kong et al. strongly suggests its involvement in pivotal developmental processes.
- WT plants VS. Transgenic plants:
- Southern blot analysis of genomic DNA from the RNAi plants confirmed that OsCCC1 was integrated into the genome of the transgenic plants and that more than half of the transgenic lines contained two or more copies of the gene. (Figure 1A)
- Real-time PCR analysis to examine the expression levels of OsCCC1in the T3 plants of the transgenic lines revealed that the expression level of OsCCC1 in RNAi transgenic lines Ri 3-4 and Ri 7-2 had decreased to 30 and 39%, respectively. (Figure 1B)
- The effect of NaCl and KCl on the germination of OsCCC1 RNAi seeds was also tested.There was no difference in seed germination between the WT and RNAi seedlings under normal conditions under normal conditions. In the presence of 150 mM NaCl, the germination of both WT and OsCCC1 RNAi seeds was inhibited slightly, but there was no obvious difference between the WT and RNAi seedlings. In the presence of KCl, the germination of OsCCC1RNAi seeds was inhibited significantly and WT seeds inhibited to a lesser extent. When the seeds were subjected to the 50 mM NaCl + 100 mM KCl treatment, approximately 96% of WT seeds germinated compared with only 83% of the seeds of the RNAi plants. Under 150 mM KCl treatment, 82% of the WT seeds germinated compared with only 41% seeds of the RNAi plants.(Figure 1C) Thus, during germination, gene silencing of OsCCC1 in rice decreased the tolerance to KCl but not to NaCl.
- The progeny of T3 homozygous kanamycin-tolerant plants (T4 generation) of lines Ri 3-4 and Ri 7-2 and WT seedlings were cultivated under salt stress (NaCl and KCl)or non-salt-stress conditions. In contrast to WT plants, the transgenic plants of line Ri 7-2 displayed progressive chlorosis, reduced leaf size, and a general growth inhibition under KCl conditions. These inhibitory effects increased progressively with increasing KCl concentrations. (Figure 1D)
- Similar phenomena occurred in the progeny of line Ri 3-4. These seedlings were harvested and their fresh and dry weight measured. Under normal conditions, the fresh and dry weight of OsCCC1 RNAi lines was lower than those of WT seedlings. The KCl and NaCl treatments significantly decreased the fresh weight and dry weight of both WT and RNAi seedlings; however, compared to normal conditions, the fresh and dry weight of RNAi seedlings decreased more obviously than those of the WT under NaCl and KCl conditions, especially under KCl conditions.Of the three stress treatments, the RNAi lines showed the greatest decrease in shoot and root fresh and dry weight compared to the WT under the 150 mM KCl treatment. (Figure 1E–H)
Expression Pattern of OsCCC1:
- The OsCCC1 transcript levels in the leaves and roots were slightly upregulated by NaCl treatment, but distinctly upregulated by KCl treatment,suggesting that OsCCC1was mainly involved in KCl rather than NaCl transport (Figure 2e, f).
- A real-time PCR analysis of OsCCC1 expression levels in different tissues revealed that OsCCC1 mRNA was more abundant in the leaves and roots, especially in leaf and root tips, than in the stem.(Figure 2g).
- OsCCC1 cDNA was obtained by PCR amplification with gene-specific primers (forward primer:5'-ATGGAGAACGGGGAGATCGAG-3'; reverse primer:5'-AAAGCTGTGAATAAAGTGGCTGAGT-3').
The full-length gene of OsCCC1 fused with C-terminal GFP (OsCCC1::GFP) was used to examine the exact subcellular localization of the OsCCC1 protein in plant cells.Two experiments designed by Xiang-Qiang Kong et al.,the results confirmed that OsCCC1 localizes to the plasma membrane of plant cells. 
- Orthologue CCC genes: A complete open reading frame (ORF) was found in the At CCC cDNA sequence, and its comparison with the genomic sequence allowed us to determine the intron/exon structure of the At CCC gene. TBLASTN searches identified orthologue CCC genes in other plant genomes. Two CCC genes were identified in rice (Oryza sativa, japonica), OsCCC1 and OsCCC2, and one more in Medicago truncatula, MtCCC.Interestingly, these plant CCC genes contained 12 introns located in the same position, indicating that the exon/intron structure was perfectly conserved.
Plant and animal CCCs were highly conserved in the C-termini and the central hydrophobic core, particularly within the putative Tm domains and the predicted intracellular loops.
- Regarding the ions involved in the symport mechanism, CCCs are divided into three groups: K+ : Cl－cotransporters, known as the KCC group; Na+ : Cl－cotransporters, NCC group; and Na+ :K+ :Cl－ cotransporters, NKCC group. Ion transport studies have demonstrated that the members of all three groups shared an absolute requirement for both Cl－ and at least one cation (Na+ and/or K+), and that the three cotransport processes are electrically silent or electroneutral.  
- When grown with unmodified soil, ccc mutants were observed to produce seed that over-accumulate Ca and S, and underaccumulate Na and K. CCC encodes the only known cation chloride co-transporter in A. thaliana. Promoter-GUS and public expression profiling analyses suggest that CCC is expressed throughout the plant, including roots, shoots, seeds and pollen.
Labs working on this gene
- Kay Lab of Plant Stress Research, School of Life Science, Shandong Normal University, 250014 Jinan, Shandong Province, People’s Republic of China
- Cotton Research Center, Shandong Academy of Agricultural Sciences, 250100 Jinan, Shandong Province, People’s Republic of China
- Kong X Q, Gao X H, Sun W, et al. Cloning and functional characterization of a cation–chloride cotransporter gene OsCCC1[J]. Plant molecular biology, 2011, 75(6): 567-578.
- Colmenero‐Flores J M, Martínez G, Gamba G, et al. Identification and functional characterization of cation–chloride cotransporters in plants[J]. The Plant Journal, 2007, 50(2): 278-292.
- McDowell S C, Akmakjian G, Sladek C, et al. Elemental concentrations in the seed of mutants and natural variants of Arabidopsis thaliana grown under varying soil conditions[J]. PloS one, 2013, 8(5): e63014.