PROLIFERATING INFLORESCENCE MERISTEM, a MADS-box gene that regulates floral meristem identity in pea

Abstract

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem (pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. The PEAM4 coding region was deleted in the pim-1 allele, and this deletion cosegregated with the pim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5' splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction of PIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.

Figure 1

Wild-type and mutant inflorescences. A, Schematic diagram of a wild-type pea plant. The center line with an arrow represents the abbreviated (//) main axis of the pea plant with its indeterminate apical meristem. At first, the apical meristem is vegetative and produces leaves (ellipses). On floral induction, the apical meristem is converted to an indeterminate primary inflorescence apex that bears secondary inflorescences (/) in the leaf axils. These in turn bear one or two flowers (●), and terminate in a stub (▾). B, Secondary inflorescence from a wild-type pea bearing two flowers. These are typical pea flowers with wild-type anthocyanin pigmentation, showing standard (st), two wings (w), and two fused petals forming the keel (k). Within the keel are the 10 stamens and a central carpel. Also visible are some of the five sepals (se), which form a cup surrounding the petals. The stub that terminates the secondary inflorescence (inf) is not visible behind the pedicel (pd). C, Young (at anthesis) secondary inflorescences from wild-type (WT), pim-1, and pim-2 flowers from plants with a white-flowered (anthocyanin absent) background. In the pim mutants, each flower is replaced by additional secondary inflorescences (inf) that bear abnormal flowers. Flowers are surrounded by leafy bracts (br) but are able to produce some petals, stamens, and carpels. Mosaic organs are also produced (mo). D, Secondary inflorescences from the same genotypes approximately 3 weeks later. A terminal stub is visible on the wild-type inflorescence (sb). The carpel of each wild-type flower has developed into a pod (p), and the proliferation of the pim mutant inflorescences has continued.

Figure 2

DNA gel-blot analysis. A, EcoRI, EcoRV, HindIII, and NcoI-digested pim-1, pim-2, and wild-type (WT) genomic DNA, probed with the C-terminal fragment of the PEAM4 cDNA and washed at low stringency. B, Ethidium bromide-stained gel of the samples shown in A, before they were blotted to a filter. Marker lane (M) contained bacteriophage lambda DNA digested with EcoRI and HindIII to generate 14 fragments, 21, 9.4, 6.6, 5.0, 4.3, 3.6, 2.3, 2.0, 1.9, 1.6, 1.4, 0.9, 0.8, and 0.6 kb in size.

Figure 3

RNA gel-blot analysis. A, Total RNA from pim-2 and wild-type (WT) flowers, probed with the C-terminal fragment of the PEAM4 cDNA. B, Total RNA from pim-2 and wild-type (WT) flowers, probed with rDNA.

Figure 4

Sequence analysis of PCR products. Alignment of sequences from PEAM4 PCR products from wild-type (WT) genomic DNA and from pim-2 and wild-type cDNA. A single guanine to adenine substitution at the 5′-splice acceptor site is highlighted in bold (arrowhead).

Figure 5

PEAM4 expression in uni and stp mutants before and after flowering. A, Northern gel blot using total RNA from sibling uni, stp, and wild-type (WT) plants, probed with the C-terminal fragment of the PEAM4 cDNA and washed at 65°C in 0.5× SSC. The first three lanes contain RNA from plants in the vegetative phase, the last three lanes contain RNA from flowering plants. The positions of the 25S and 18S ribosomal RNA bands are shown on the right. The PEAM4 transcript is indicated (arrow). B, Ethidium bromide-stained gels of the samples shown in A, before they were blotted to a filter.

Figure 6

RNA in situ analysis of PEAM4 expression in developing pea flowers. A, PEAM4 expression in wild-type genotype HL 107 was confined to the flower and was not detected in vegetative or inflorescence tissue (I1 and I2 indicate the primary and secondary inflo- rescences, respectively). Expression occurred throughout young floral primordia (flower F1) at stage 2 and was also apparent in the oblique section through flower 2 (F2) and in a transverse section through the base of an older flower bud (FB) at approximately stage 7 of development. B, Stage 4 (F1) of development in genotype HL 107, showing PEAM4 expression in the petal region of the petal/stamen common primordia (pe) and the sepals (se). Expression was absent from the stamen region of the petal/stamen common primordia (st) and the carpel primordium (c). Stage 5 (F2) flower showing expression in the outer sepal whorl. Petals express PIM at this stage but were not in the plane of this section. C, Stage 7 flower bud of genotype HL 107 showing PEAM4 expression limited to sepals and petals and absent from stamens (st) and the carpel. D, Stage 7 flower bud of the afila genotype, JI 1195, showing PIM expression limited to the two outer whorls and absent from the subtending compound leaf (L). Magnification ×75 in A through D.