The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development

Abstract

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.

Figure 1.

Phenotype of dad1 Alleles. (A) Wild-type V26, dad1-1, dad1-2, and dad1-3 plants after 12 weeks of growth. (B) Quantitative phenotypic analysis of V26 and dad1 mutant alleles. Plants were grown for 12 weeks as described in Methods. Number of branches, overall height, the node of the highest branch, and the number of nodes to the first flower were measured for the wild type, dad1-1 (d1-1), dad1-2 (d1-2), and dad1-3 (d1-3; n = 16 for each genotype).Mean values ± se are shown. (C) Branch distribution within wild-type and dad1 alleles. The colored bars represent the proportion of plants (n = 16 for each genotype) that had a branch at that node. The value for the cotyledonary node (cot) is >1.0 when there is a branch at >1 cotyledonary axil (1 node) for the majority of the plants. (D) Scanning electron micrographs of V26 and dad1-1 plants. Seedlings were germinated in vitro and grown until the first true leaves were ∼2 mm in length. The cotyledonary axils are shown. The gray scale bars in each photograph are 0.1 mm in length. S, main shoot; C, cotyledon. Arrowhead on the wild type (V26) indicates axillary meristem.

Figure 2.

Structure of the Dad1/PhCCD8 Gene. (A) Structure of the Dad1/PhCCD8 gene showing the locations of the mutant alleles. Open boxes represent the ORF within the exons, with introns represented by thin black lines. The promoter and 5′ and 3′ UTRs are denoted by shaded bars. The sequence associated with the dad1-1 allele represents the Dad1/PhCCD8 sequence that was duplicated by the insertion of a transposon (the actual transposon sequence is not shown), and the sequence associated with the dad1-3 allele is the 9-bp footprint left by the excision of the transposon. For dad1-2, the single base change is shown. (B) Alignment of the predicted amino acid sequence of the Dad1 wild-type and mutant alleles around the position of the insertion in dad1-1, in comparison with other members of the CCD8 clade. The consensus sequence shows amino acids that are absolutely conserved in the sequences shown (the mutant alleles were not used in consensus generation). The differences between the wild-type Dad1 sequence and the mutant alleles are underlined. In dad1-1, the asterisk indicates a stop codon. (C) Alignment of the Dad1 and dad1-2 predicted amino acid sequence in comparison with one member of each clade of the CCD gene family. The consensus shows amino acids for which at least three members have the sequence shown (the dad1-2 sequence was not used in the consensus generation). The changed amino acid in the dad1-2 allele is underlined. (D) Repeat structure of the transposon (Dot) inserted in the dad1-1 allele, including the 10-bp direct duplication of the Dad1 sequence. The key shows the consensus sequence for each motif found in the transposon sequence with the maximum number of mismatches allowed for inclusion in the figure. The longer CT-rich region is composed of 93% CT and the shorter region is 100% CT, whereas the GA-rich region is 89% GA. (E) Unrooted phylogenetic tree of the CCD gene family. Only full-length members of the family are included, and not all members in the NCED clade are included for clarity (though all known Arabidopsis members of the gene family are included). The predicted protein sequences were initially clustered using ClustalX (Thompson et al., 1997) and the alignment refined manually with the program MacClade (Maddison and Maddison, 2000). Phylogenetic relationships between the family members were determined using the neighbor-joining method and bootstrap analysis with 1000 replicates using the Phylip package of programs (Felsenstein, 2004). Bootstrap values are shown as percentages. Accession numbers for sequences used from the following species are as follows: Arabidopsis AtCCD1 (At3g63520), AtNCED2 (At4g18350), AtNCED3 (At3g14440), AtCCD4 (At4g19170), AtNCED5 (At1g30100), AtNCED6 (At3g24220), AtCCD7 (At2g44990), AtCCD8 (At4g32810), and AtNCED9 (At1g78390); Bixa orellana BoLCD (AJ489277.1); Crocus sativus CsZCD (CAD33262.1); Zea mays ZmVP14 (U95953.1); rice OsCCD7 (AL663000.4), OsCCD8a (AP003296.3), and OsCCD8b (AP003376.3); petunia Dad1/PhCCD8 (AY743219); pea RMS1 (AY557341.1); Sphingomonas paucimobilis PpLSD1 (S80637.1), PpLSD2 (S65040.1), and PpLSD3 (AB073227.1); Streptomyces antibioticus SaSIM14 (AF322256.1); Synechocystis sp SynLSD1 (D90914.1) and SynLSD2 (AP005369.1); Anabaena sp AnaLSD (AP003595.1); Homo sapiens HsRPE65 (AF039855.1), HsCCD (AJ290393.1), and HsCCD3 (AF294900.1); Drosophila melanogaster DmCCD (AY121617.1); Caenorhabditis elegans CeCCD3 (AL110485.5) and CeCCD4 (AF098992.2).

Figure 3.

Complementation and Phenocopying of the dad1-1 Mutant Phenotype. (A) Complementation of dad1-1 with Dad1/PhCCD8 and phenocopying of the mutant phenotype in V26 with a Dad1/PhCCD8 RNAi construct. Plants of similar ages with a control transgene are shown for comparison. (B) Branching phenotypes of control (MD) and three independent Dad1/PhCCD8 RNAi lines 8 weeks postgermination. (C) The same plants as shown in (B) 12 weeks postgermination. (D) Plants from (C) shown stripped of their leaves.

Figure 4.

Expression of the Dad1/PhCCD8 Gene in Petunia. (A) Dad1/PhCCD8 expression in roots and leaves of Dad1/PhCCD8 RNAi plants as determined by TaqMan real-time RT-PCR. (B) Dad1/PhCCD8 expression in the tissues of wild-type MD plantsas determined by TaqMan real-time RT-PCR. Roots were taken from12-week-old plants separated into tip, middle, and base sections and analyzed independently. Stem tissue included the entire stem and nodes from mature flowering plants ranging from 30 to 35 cm in height from the base to the flower. (C) Real-time RT-PCR analysis of Dad1/PhCCD8 transcripts in 7-week-old V26 and dad mutant plants. The shoot apex sample included the apex of the plant, including leaves smaller than 1.5 cm in length. The root and stem samples included all root or stem tissue (except for that included in the shoot apex samples) from a pool of six plants. The plants were not flowering at the time of harvest.

Figure 5.

Flowering-Associated Phenotypes in Dad1/PhCCD8 Loss-of-Function Lines. (A) Number of nodes before the terminal flower in Dad1/PhCCD8 RNAi lines and dad1-1 compared with wild-type controls (MD and V26). (B) Flower weight of Dad1/PhCCD8 RNAi lines and dad1-1 compared with wild-type controls. (C) Flowers from Dad1/PhCCD8 RNAi lines and dad1-1 compared with the wild type.

Figure 6.

Root Phenotypes Associated with Loss of Dad1/PhCCD8. (A) Roots of wild-type (MD) and Dad1/PhCCD8 RNAi plants 12 weeks postgermination. (B) Roots of wild-type (V26) and dad1-1 plants 13 weeks postgermination. (C) The development of roots on cuttings from MD and Dad1/PhCCD8 RNAi lines after 6 weeks. (D) Adventitious roots developing on the lower stems of a Dad1/PhCCD8 RNAi line but not on wild-type (MD) stems.