Graft transmission of a floral stimulant derived from CONSTANS

Abstract

Photoperiod in plants is perceived by leaves and in many species influences the transition to reproductive growth through long-distance signaling. CONSTANS (CO) is implicated as a mediator between photoperiod perception and the transition to flowering in Arabidopsis. To test the role of CO in long-distance signaling, CO was expressed from a promoter specific to the companion cells of the smallest veins of mature leaves. This expression in tissues at the inception of the phloem translocation stream was sufficient to accelerate flowering at the apical meristem under noninductive (short-day) conditions. Grafts that conjoined the vegetative stems of plants with different flower-timing phenotypes demonstrated that minor-vein expression of CO is able to substitute for photoperiod in generating a mobile flowering signal. Our results suggest that a CO-derived signal(s), or possibly CO itself, fits the definition of the hypothetical flowering stimulant, florigen.

Figure 1.

Accelerated flowering and graft transmission of floral stimulant resulting from CO expression confined to the companion cells of minor veins in mature leaves. A, T1 generation plants (Columbia ecotype) transformed with an empty-vector control (left) and GAS:CO-containing vector (right) grown for 41 d with 10-h-light/14-h-dark cycles. B, Vegetative stem section of grafted plant 10 d after grafting. Arrow points to callus tissue formed between the scion (top) and stock (bottom); scale bar = 0.5 mm. C, Scion homozygous for the co-1 allele showing floral buds 20 d after grafting to a GAS:CO stock plant. D, Scion homozygous for the co-1 allele showing a vegetative apex 20 d after grafting to a homozygous co-1 allele stock plant. Flowers were not visible until 43 d after grafting. Scale bar for C and D = 2 mm.

Figure 2.

RT-PCR analysis of CO mRNA distribution. Total RNA was extracted from a wild-type plant (lane 1) and different tissues of a co-1 scion grafted to a GAS:CO stock plant. Lane 2, Homozygous co-1 scion inflorescence; lane 3, homozygous co-1 scion mature leaf; lane 4, GAS:CO stock axillary inflorescence; lane 5, GAS:CO stock mature leaf. A, RT-PCR with a primer pair specific for GAS:CO transcripts. B, RT-PCR with a primer pair specific for wild-type CO transcripts. C, RT-PCR with a primer pair specific for co-1 allele transcripts.

Figure 3.

X-glcA staining in GAS:T7CO-GUS plants demonstrating promoter specificity. A, Expression is confined to the minor veins of mature leaves. Note absence of staining in the midrib and petiole. X-glcA staining was for 24 h in the presence of 0.1 mm of each potassium ferricyanide and potassium ferrocyanide; scale bar = 0.5 mm. B, T7CO-GUS fusion protein localized to the nuclei of companion cells in minor veins. Pictured is a blind-ending vein. X-glcA staining was for 24 h in the presence of 2.0 mm of each potassium ferricyanide and potassium ferrocyanide; scale bar = 0.05 mm. C, Absence of X-glcA staining in the vegetative body and apical meristem, demonstrating the absence of gene expression from GAS promoter in these regions; scale bar = 1 mm. B and C are from the same stained plant, while A is a sibling grown in the same pot. These staining patterns are characteristic of those observed in 15 independent transformants. D, Overlay (right) of X-glcA staining (left) with DAPI-stained nuclei (center) of a branched minor vein (outlined); arrows indicate overlapping X-glcA and DAPI stain. X-glcA staining was for 24 h in the presence of 5.0 mm of each potassium ferricyanide and potassium ferrocyanide; scale bar = 0.02 mm.