The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression

Abstract

AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunochemistry, we found that the AGL15 protein accumulates transiently in the shoot apex in young Arabidopsis and Brassica seedlings and that promoter activity is associated with the shoot apex and the base of leaf petioles throughout the vegetative phase. During the reproductive phase, AGL15 accumulates transiently in floral buds. When AGL15 was expressed in Arabidopsis under the control of a strong constitutive promoter, we noted a striking increase in the longevity of the sepals and petals as well as delays in a selected set of age-dependent developmental processes, including the transition to flowering and fruit maturation. Although ethylene has been implicated in many of these same processes, the effects of AGL15 could be clearly distinguished from the effects of the ethylene resistant1-1 mutation, which confers dominant insensitivity to ethylene. By comparing the petal breakstrength (the force needed to remove petals) for flowers of different ages, we determined that ectopic AGL15 had a novel effect: the breakstrength of petals initially declined, as occurs in the wild type, but was then maintained at an intermediate value over a prolonged period. Abscission-associated gene expression and structural changes were also altered in the presence of ectopic AGL15.

Figure 1.

Constructs Used for Arabidopsis Transformations. (A) Reporter construct (pAGL15:GUS). Genomic sequences (hatched rectangles) flanking the AGL15 coding sequence (5′ and 3′) were fused to the coding sequence for GUS. (B) Construct for constitutive expression of AGL15 (p35S:AGL15). The promoter of the 35S gene of the cauliflower mosaic virus was fused to the AGL15 cDNA. (C) Construct for constitutive expression of AGL15 (p35S:gAGL15). Similar to the construct shown in (B), p35S:gAGL15 includes the first three introns (black rectangles) of the AGL15 gene. NOS, nopaline synthase; arrowheads, transcription start/stop; bent arrows, translation start; black circles, translation stop.

Figure 2.

Analysis of AGL15 Promoter Activity in Transgenic Arabidopsis Plants Carrying AGL15 Promoter:GUS Reporter Constructs. GUS activity appears as blue staining in bright-field images and as pink crystals in dark-field images. (A) Section of developing seed, stained with toluidine blue to show internal tissues. (B) Dark-field microscopy showing GUS activity in developing seed. (C) GUS activity is associated with the shoot apex (arrowhead) in a 7-day-old seedling. (D) Section of the shoot apex of an 8-day-old seedling stained with toluidine blue. (E) Dark-field microscopy showing GUS activity in the meristem, leaf primordia, stipules, and trichome initials (arrowhead) in an 8-day-old seedling. (F) GUS activity in the shoot apex (arrowhead) of a plant growing vegetatively under noninductive (short-day) conditions. (G) Section of the shoot apex of a plant growing under short-day conditions, stained with toluidine blue to show the shoot apical meristem and associated structures. (H) Dark-field microscopy showing GUS activity in the shoot apical meristem, axillary meristems (arrowheads), stipules, and young leaf primordia of a plant grown under short-day conditions. (I) and (J) GUS activity associated with the base of the cauline leaves (arrowheads) in the inflorescence. (K) GUS activity associated with young floral buds (black arrowhead) and with the basal part of older floral buds (white arrowhead). (L) Dark-field microscopy showing GUS activity in the apex of an inflorescence. No GUS activity is detectable in the inflorescence meristem or youngest floral buds. (M) Dark-field microscopy showing GUS activity associated with cells in the receptacle and at the base of the floral organs in a stage 11 floral bud. (N) GUS activity in floral buds of different ages. GUS activity declines with increasing age and cannot be detected in buds that are ready to open (arrowheads). am, axillary meristem; an, anther; c, cotyledon petiole; ca, carpel; cl, cauline leaf; e, embryo; en, endosperm; fb, floral bud; l, leaf; lb, leaf buttress; m, meristem; pe, pedicel; re, receptacle; sc, seed coat; se, sepal; sp, septum of fruit; st, stipule primordium. .

Figure 3.

Analysis of AGL15 mRNA and Protein Accumulation in Arabidopsis and Brassica. (A) Protein gel blot of soluble protein extracts (150 μg per lane) from various Arabidopsis tissues probed with affinity-purified anti-AGL15 antibodies. The inflorescence apex sample included all of the tissues within 1 cm of the youngest buds. (B) Immunolocalization of AGL15 in the shoot apex of a 4-day-old Arabidopsis seedling. AGL15 is associated with nuclei in the shoot apical meristem and in leaf primordia. . (C) AGL15 mRNA accumulation in various Brassica tissues. A slot blot loaded with equal amounts of tRNA or poly(A)⁺ RNA was hybridized with an AGL15-specific probe prepared from a sequence downstream of the MADS box. Samples labeled abscission zone/receptacle (AZ/R) were prepared from the basal regions of open flowers at anthesis or from the basal regions of developing fruits. (D) Protein gel blot of soluble protein extracts (75 μg per lane) from young Brassica floral buds or the basal portions (AZ/Receptacle) of older floral buds, flowers, or developing fruits, probed with affinity-purified anti-AGL15 antibodies. (E) Immunolocalization of AGL15 in the basal region of an immature (3 to 4 mm) Brassica floral bud. . (F) Immunolocalization of AGL15 in the basal region of a mature Brassica flower. . an, anther; c, cotyledon; ca, carpel; DAP, days after pollination; Infl. apex, inflorescence apex; l, leaf; m, meristem; n, nectary; o, ovule; p, petal; re, receptacle; se, sepal.

Figure 4.

Analysis of Effects of Constitutive Expression of AGL15 in Arabidopsis. (A) Protein gel blot of soluble protein extracts (150 μg per lane) from leaves of wild-type or transgenic Arabidopsis plants incubated with anti-AGL15 affinity-purified antibodies. Plants carrying the intron-bearing construct (p35S:gAGL15) accumulated more AGL15 than did plants carrying the cDNA construct (p35S:AGL15). (B) Immunolocalization of AGL15 in the apex of the inflorescence of a wild-type Arabidopsis plant. (C) Immunolocalization of AGL15 in the apex of the inflorescence of a transgenic Arabidopsis plant carrying the p35S:gAGL15 construct. (D) Vegetative rosette of a wild-type (Ws ecotype) plant grown for 19 days under long-day conditions. (E) Vegetative rosette of a transgenic plant that expresses AGL15 constitutively, grown for 33 days under long-day conditions. (F) Inflorescence of a wild-type (Ws ecotype) plant. (G) Inflorescence of a transgenic plant that expresses AGL15 constitutively. (H) Petals and sepals (arrowhead) associated with developing fruits on transgenic plants that express AGL15 constitutively. (I) Developing embryo at 7 DAP in seed of a wild-type plant. (J) Developing embryo at 7 DAP in seed of a transgenic plant that expresses AGL15 constitutively. (K) Abscission zone associated with the sepal of a wild-type plant. (L) Abscission zone associated with the sepal of a transgenic plant that expresses AGL15 constitutively. az, abscission zone; cl, cauline leaf; e, embryo; fb, floral bud; m, meristem; pe, pedicel; re, receptacle; se, sepal. ; ; .

Figure 5.

Analysis of Abscission in Wild-Type Plants and Transgenic Arabidopsis Plants That Express AGL15 Constitutively. (A) Comparison of petal breakstrength in wild-type plants and plants that express AGL15 constitutively. A stress transducer was used to measure the force required to remove petals from flowers at different positions along the main inflorescence axis. Position 1 corresponds to the apical-most flower with visible white petals. Higher numbers correspond to more basal positions and successively older flowers. Bars indicate the standard error of the mean. For positions 2, 4, and 6, ; for other positions, n ⩾ 10 flowers. (B) to (E) Scanning electron microscopy of petal abscission zones in wild-type plants. Petals were forcibly removed from flowers in positions 3 (B) and 5 (C). Petals had abscised naturally from flowers in positions 7 (D) and 9 (E). (F) to (I) Scanning electron microscopy of abscission zones in plants that express AGL15 constitutively (p35S:AGL15). Petals were forcibly removed from flowers in positions 3 (F), 5 (G), 7 (H), and 9 (I). (J) GUS activity in flowers at position 6 on Arabidopsis plants carrying the pBAC (bean abscission cellulase):GUS reporter construct. The plant on the right carried the p35S:gAGL15 construct and constitutively expressed AGL15. The plant on the left was a sibling that did not carry the AGL15 construct. .