The CYCLIN-DEPENDENT KINASE Module of the Mediator Complex Promotes Flowering and Reproductive Development in Pea

Abstract

Control of flowering time has been a major focus of comparative genetic analyses in plant development. This study reports on a forward genetic approach to define previously uncharacterized components of flowering control pathways in the long-day legume, pea (Pisum sativum). We isolated two complementation groups of late-flowering mutants in pea that define two uncharacterized loci, LATE BLOOMER3 (LATE3) and LATE4, and describe their diverse effects on vegetative and reproductive development. A map-based comparative approach was employed to identify the underlying genes for both loci, revealing that that LATE3 and LATE4 are orthologs of CYCLIN DEPENDENT KINASE8 (CDK8) and CYCLIN C1 (CYCC1), components of the CDK8 kinase module of the Mediator complex, which is a deeply conserved regulator of transcription in eukaryotes. We confirm the genetic and physical interaction of LATE3 and LATE4 and show that they contribute to the transcriptional regulation of key flowering genes, including the induction of the florigen gene FTa1 and repression of the floral repressor LF Our results establish the conserved importance of the CDK8 module in plants and provide evidence for the function of CYCLIN C1 orthologs in the promotion of flowering and the maintenance of normal reproductive development.

Figure 1.

Mutations at LATE3 and LATE4 loci delay flowering and prolong the reproductive phase. A, Representative wild-type (WT; NGB5839), late3, and late4 plants grown under 16-h long-day conditions. To account for the disparity in flowering time, this image compares WT and mutants at 62 and 130 d after sowing, respectively. B and C, Effect of photoperiod and vernalization on flowering initiation in WT, late3, and late4, mutant plants. Data represent mean ± se for n = 6 to 8 plants. +V, vernalization; NFI, node of flower initiation; I-RN, initial reproductive nodes; IR-VN, inflorescence reverted-vegetative nodes; l-RN, later reproductive nodes.

Figure 2.

Mutations at LATE3 and LATE4 loci affect varied aspects of reproductive development. A, Total number of flowers and pods at maturity. B, Seed content per pod (from one to five seeds) expressed as a proportion of the total number of seed-bearing pods. C, 10-seed dry weight and (D) total number of seeds per plant. Data were collected at the time of harvest and represented as mean ± se for n = 6 (A, B, and D) or n = 3 (C).

Figure 3.

Mutant alleles at LATE3 and LATE4 loci carry mutations in genes encoding Mediator complex components CYCLIN-DEPENDENT KINASE8 and CYCLIN C1, respectively. Diagrams showing (A) gene structure of PsCDK8 and the nature and location of mutations in late3 alleles and (B) gene structure of PsCYCC1 and the nature and location of mutations in late4 alleles. Exons are represented by numbered boxes, with gray shading designating 5′ and 3′ UTRs. Dashed lines in A represent introns not fully characterized. Sequence details of mutations and splice variants are shown in Supplemental Figs. S9, S12, and S13.

Figure 4.

LATE3 and LATE4 show genetic and physical interaction. A to C, Comparison of wild type, late3-1, and late4-2 single mutants and the late3-1 late4-2 double mutant grown under LD conditions. A, Representative 75-d-old plants. B, Node of flower initiation. C, Representative leaflet area (single leaflet from leaf 10). Data represent mean ± se for n = 6 to 10 plants. D, Yeast two-hybrid analysis for interaction between PsCDK8 and PsCYCC1 proteins from wild-type (NGB5839) genotype. The image shows diploid yeast colonies derived via mating of haploid yeast strains PJ694 alpha and PJ694 A carrying different bait and prey plasmids for experimental and control interactions (as indicated). For each interaction tested, two colonies derived from independent matings (top, colony 1; bottom, colony 2) were used grown in selective interaction-specific (SC-L-W-H +10 mm 3A-amino-1,2,4-triazole [3AT], right) and selective mating-specific (SC-L-W, left) medium and incubated at 30°C for 4 d. Key interactions are highlighted in red.

Figure 5.

Genetic interactions of late3 and late4 mutants with early-flowering mutants lf and sn. A, Node of flower initiation (NFI) and number of reproductive nodes (RN) in wild-type, lf, late3-2, late4-1, lf late3-2, and lf late4-1 genotypes. B, Node of flower initiation in wild-type, sn-4, late4-1, and sn-4 late4-1 genotypes. C and D, Representative plants at 75 (C) and 63 d (D) after sowing. All plants were grown in long days. Data in A and B represent mean ± se for n = 6 to 10 plants.

Figure 6.

LATE3 and LATE4 loci affect expression of several florigen family and inflorescence-identity genes. Developmental time courses for expression of key flowering genes in leaf and shoot apex material from wild type NGB5839 (black), late3-1 mutant (blue), and late4-2 mutant (red) grown under long-day conditions. Data have been normalized to the reference gene TFIIa and represent mean ± se for n = 3 biological replicates, each consisting of material pooled from two different plants. Black and blue dashed lines indicate the time that flower buds first became visible in dissected shoot apices of wild type and late3-1 mutants, respectively. Flower initiation did not occur in late4-1 mutants for the duration of the experiment. Time points at which expression in wild type was significantly different from both mutants (P ≤ 0.05) are indicated with an asterisk.