The Cytochrome P450 Gene CsCYP85A1 Is a Putative Candidate for Super Compact-1 (Scp-1) Plant Architecture Mutation in Cucumber (Cucumis sativus L.)

Abstract

The dwarf or compact plant architecture is an important trait in plant breeding. A number of genes controlling plant height have been cloned and functionally characterized which often involve in biosynthesis or signaling of plant hormones such as brassinosteroids (BRs). No genes for plant height or vine length have been cloned in cucurbit crops (family Cucurbitaceae). From an EMS-induced mutagenesis population, we identified a super compact (SCP) mutant C257 which was extremely dwarf due to practically no internode elongation. Under dark growing condition, C257 did not undergo skotomorphogenesis and its mutant phenotype could be rescued with exogenous application of brassinolide (BL), suggesting SCP might be a BR-deficient mutant. Segregation analysis revealed a single recessive gene scp-1 that was responsible for the SCP mutation. Map-based cloning combined with a modified MutMap identified CsCYP85A1, a member of the plant cytochrome P450 monooxygenase gene family, as the most possible candidate gene for scp-1, which encodes a BR-C6-oxidase in the BR biosynthesis pathway. We show that a SNP within the second exon of scp-1 candidate gene caused the SCP phenotype. Three copies of the CsCYP85A gene are present in the cucumber genome, but only the scp-1/CsCYP85A1 gene seemed active. The expression of CsCYP85A1 was higher in flowers than in the leaves and stem; its expression in the wild type (WT) was feedback regulated by BL application. Its expression was reduced in C257 as compared with the WT. This was the first report of map-based cloning of a plant height gene in cucurbit crops. The research highlighted the combined use of linkage mapping, an improved MutMap method and allelic diversity analysis in natural populations in quick cloning of simply inherited genes in cucumber. The roles of CsCYP85A1 in regulation of internode elongation in cucumber was discussed.

Keywords: BR biosynthesis; CYP85A1; cucumber; cytochrome P450; dwarf; plant architecture.

FIGURE 1

Morphological characterization of WT (CCMC) and C257 mutant. (A,B) Seventy-day-old plants of CCMC and C257, respectively. Longitudinal and cross sections of CCMC (C,E) and C257 (D,F) stems reveal shorter and wider cells of C257 than that of the WT. VB, vascular bundles; PC, parenchyma cells; Bar = 5 cm (A,B); Bar = 500 μm (C,D); Bar = 1 mm (E,F).

FIGURE 2

Skotomorphogenic and photomorphogenic responses of WT and C257 SCP mutant. The WT seedling exhibits typical skotomorphogenic (A, left, in dark) and photomorphogenic (B, left, under light) responses whereas C257 mutant has little hypocotyls elongation and open cotyledons in the dark (A, right). Hypocotyl length data in (C, D) were collected at 10 days after germination. ^∗∗Indicate statistically significant differences of expression level based on Duncan’s test (P < 0.05). Bar = 1 cm.

FIGURE 3

Responses to BL application in C257 mutant. At cotyledon stage, 100 μM BL was sprayed on C257 seedling plant. As compared with the control (water mock spray) (A,C,E), BL application can partially rescue the mutant phenotype as seen from the plant height (B), leaf growth (D), and elongation of internodes (F). Bar = 5 cm.

FIGURE 4

Map-based cloning of scp-1 locus. Initial framework mapping with 46 F₂ plants identified 11 SSR markers linked with scp-1 in cucumber Chromosome 5 (A). Linkage analysis with new markers in 184 F2 plants delimits the scp-1 locus between SSR31415 and UW050632 (B). SNP index plot shows possibly harboring causal mutations in chromosome 5 for C257 using MutMap. Red regression lines were obtained by averaging SNP indices from a moving window of five consecutive SNPs and shifting the window one SNP at a time. The x-axis value of each averaged SNP index was set at a midpoint between the first and fifth SNP (C). Fourteen SNPs located in eight gene exon region were extracted. Green swallowtails and black arrows indicate genes and mutation sites, respectively (D). The candidate gene encodes a putative cytochrome P450 protein CsCYP85A1 which has nine exons and eight introns. The SNP in the second exon results in a tryptophan codon at 157 position (TGG) substitution to a stop codon (TAG). Blue rectangles with consecutive numbers and black line indicate exons and introns, respectively. Mutation identified in C257 are indicated (E).

FIGURE 5

Phylogenetic tree of CYP85A protein in cucumber and its homologs in 10 other species. The phylogenetic tree was generated using the neighbor-joining method built in MEGA 7.0, and the inferred phylogeny was tested by bootstrap analysis with 1000 replicate datasets. Numbers shown at the tree forks indicate frequency of occurrence among all bootstrap iterations performed.

FIGURE 6

Expression level of the CsCYP85A1 candidate gene in WT and C257 plants with BL treatment revealed from semi-quantitative PCR (A,C,E) and qPCR (B,D,F). The expression of scp-1/CsCYP85A1 in WT plants was dramatically reduced by treatment with 100 μM BL (A,B), which was the same in various organs at flowering stage (C,D). Exogenous BL treatment did not alter scp-1/CsCYP85A1 expression in the mutant C257 (A,B,E,F). ^∗∗Indicates statistically significant differences of expression level based on t-test (P < 0.01). R, S, L, F, and M represents root, stem, leaf, female flower, and male flower, respectively.