Isolation, Characterization and Transcriptome Analysis of a Cytokinin Receptor Mutant Osckt1 in Rice

Abstract

Cytokinins play important roles in regulating plant development, including shoot and root meristems, leaf longevity, and grain yield. However, the in planta functions of rice cytokinin receptors have not been genetically characterized yet. Here we isolated a rice mutant, Osckt1, with enhanced tolerance to cytokinin treatment. Further analysis showed that Osckt1 was insensitive to aromatic cytokinins but responded normally to isoprenoid and phenylurea-type cytokinins. Map-based cloning revealed that the mutation occurred in a putative cytokinin receptor gene, histidine kinase 6 (OsHK6). OsCKT1 was found to be expressed in various tissues throughout the plant and the protein was located in the endoplasmic reticulum. In addition, whole-genome gene expression profiling analysis showed that OsCKT1 was involved in cytokinin regulation of a number of biological processes, including secondary metabolism, sucrose and starch metabolism, chlorophyll synthesis, and photosynthesis. Our results demonstrate that OsCKT1 plays important roles in cytokinin perception and control of root development in rice.

Keywords: Oryza sativa; cytokinin; histidine kinase; rice; transcriptome.

FIGURE 1

Phenotypic characterization of the wild-type (WT) and Osckt1. (A) Growth of 8-day-old seedlings of the WT and Osckt1 without or with 0.2 μM 6-Benzylaminopurine (BA) treatment. (B) Enlarged view of roots of the WT and Osckt1 from (A). (C) Effect of BA treatment on lateral root number, shoot height, and primary root length of the WT and Osckt1. Significant differences were determined using Student’s t-test (^∗P < 0.05). (D) Root growth of 8-day-old WT and Osckt1 under treatments of 0.2 μM KT, 0.05 μM tZ, 0.1 μM iP, 0.02 μM CPPU, and 0.02 μM TDZ. Bars = 2 cm.

FIGURE 2

Map-based cloning of OsCKT1. (A) OsCKT1 was mapped to a 2137 kb region on chromosome 2. The rates of recombinants in the F₂ population are listed below the molecular markers. (B) Gene structure of OsCKT1. Black boxes represent exons, and lines indicate introns and the 5′ and 3′ untranslated regions. The point mutation in the last exon is indicated. (C) Secondary structure analysis of OsCKT1 using the CDD database (https://www.ncbi.nlm.nih.gov/cdd). Black boxes represent transmembrane domains. The point mutation results in a substitution of Proline by Serine in the REC domain. (D) Protein sequence alignment of the REC domain of HKs from Arabidopsis and rice. Identical and highly similar amino acids are highlighted in black and framed in black by ESPript, respectively. The mutated residue in Osckt1 was indicated by asterisk. (E–G) Complementation analysis of the Osckt1 mutant. Two independent lines of over-expression transgenic plants (Ov1 and Ov2) in the Osckt1 mutant background were displayed (E) and the enlarged view of roots under BA treatment was shown (F). RT-PCR analysis (G) of OsCKT1 in roots of Ov1 and Ov2. Bar = 2 cm.

FIGURE 3

Expression pattern of OsCKT1 and subcellular localization of OsCKT1. (A–J) Promoter-β-glucuronidase (GUS) fusion studies reveal the expression of OsCKT1 in various tissues, root tip (A), lateral root primordium (B), emerging lateral roots (C), lateral root tips (D), leaf (E), stem, ligule, and auricle (F), young spikelet (G), glume (H,I), and flower (J). (K) OsCKT1 targets green fluorescent protein (GFP) to ER in transiently transformed onion epidermal cells. The PHF1-RFP is used as the endoplasmic reticulum (ER) marker. (A,C–F,H,J) Bars=0.5 mm; (B,G,I) Bars=0.2 mm; (K) Bar = 20 μm.

FIGURE 4

Analysis and gene ontology (GO) enrichment of differentially expressed genes (DEGs) between the WT and Osckt1 under BA treatment by RNA-seq. (A) General information of sequencing reads and mapping. (B) The number of up- and down-regulated DEGs between the WT and Osckt1. (C–E) GO term enrichment analysis of up- and down-regulated DEGs in Biological process (C), Molecular function (D), and Cellular component (E).

FIGURE 5

Pathway enrichment and MapMan overview of DEGs between the WT and Osckt1 under BA treatment. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of up- and down-regulated DEGs. (B) Overview of all DEGs involved in metabolic processes by MapMan.

FIGURE 6

Expression of DEGs between the WT and Osckt1 under BA treatment associated with the Calvin cycle and Starch and Sucrose metabolism. (A–C) DEGs associated with the Calvin cycle (A), sucrose (B), and starch metabolism (C) are shown, with a table showing gene names, putative functions, and fold change. The values in red and blue indicate log₂-transformed fold increase and decrease in expression, respectively.