Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis ZINC FINGER PROTEIN2

Abstract

Organ detachment requires cell separation within abscission zones (AZs). Physiological studies have established that ethylene and auxin contribute to cell separation control. Genetic analyses of abscission mutants have defined ethylene-independent detachment regulators. Functional genomic strategies leading to global understandings of abscission have awaited methods for isolating AZ cells of low abundance and very small size. Here, we couple laser capture microdissection of Arabidopsis thaliana stamen AZs and GeneChip profiling to reveal the AZ transcriptome responding to a developmental shedding cue. Analyses focus on 551 AZ genes (AZ(551)) regulated at the highest statistical significance (P < or = 0.0001) over five floral stages linking prepollination to stamen shed. AZ(551) includes mediators of ethylene and auxin signaling as well as receptor-like kinases and extracellular ligands thought to act independent of ethylene. We hypothesized that novel abscission regulators might reside in disproportionately represented Gene Ontology Consortium functional categories for cell wall modifying proteins, extracellular regulators, and nuclear-residing transcription factors. Promoter-beta-glucuronidase expression of one transcription factor candidate, ZINC FINGER PROTEIN2 (AtZFP2), was elevated in stamen, petal, and sepal AZs. Flower parts of transgenic lines overexpressing AtZFP2 exhibited asynchronous and delayed abscission. Abscission defects were accompanied by altered floral morphology limiting pollination and fertility. Hand-pollination restored transgenic fruit development but not the rapid abscission seen in wild-type plants, demonstrating that pollination does not assure normal rates of detachment. In wild-type stamen AZs, AtZFP2 is significantly up-regulated postanthesis. Phenotype data from transgene overexpression studies suggest that AtZFP2 participates in processes that directly or indirectly influence organ shed.

Figure 1.

Developmental stages of Arabidopsis flowers and scanning electron and light micrographs of floral organ AZs. A, Flower stages. Scale bar, 1 mm. B, SEM of stamen AZ fracture planes. Scale bar, 20 μm. C, SEM of proximal faces of AZs from stamens (StAZ), sepals (SAZ), and petals (PAZ). Scale bar, 100 μm. D and E, Sectioned floral tissue before (D) and after (E) LCM of stamen AZs (shown within boxes).

Figure 2.

Classification of 551 AZ transcripts into eight clusters with similar patterns of gene expression. Clustering algorithms are described in “Materials and Methods”. [See online article for color version of this figure.]

Figure 3.

Expression profiles of selected cell separation regulators represented in AZ₅₅₁. Mean natural log signal intensities are plotted across developmental stages. Gene names are defined in the text.

Figure 4.

Reporter gene expression in plants harboring AtZFP2 promoter-GUS gene fusion. A, Promoter-GUS expression in transgenic inflorescences. B, AZ scars following floral organ detachment. C, AZ scars later in silique development. D, Stamen. E, Carpel. F, Two-week-old seedling. G, Inflorescence. H, Magnified view of leaf surface of G. I, Magnified view of stem surface of G. Scale bars, 1,000 μm.

Figure 5.

Floral organ abscission in wild-type and 35S∷AtZFP2 plants. A, Inflorescence morphology in wild-type plants. B, Transgenic line with mild phenotype. C and E, Transgenic lines with strong phenotypes. D, Transgenic line with severe phenotype. F to I, Reproductive structures from plants in A to D, respectively. J to L, Floral organ abscission responses after cross-pollination with wild-type pollen grains. J, Mild transgenic line 4 d postpollination. K, Strong transgenic line 12 d postpollination. L, Severe transgenic line 32 d postpollination. Scale bars, 1 mm.

Figure 6.

SEMs of wild-type and 35S∷AtZFP2 floral organ AZ fracture planes. Organs abscised naturally or were manually removed at designated positions along plant inflorescences. Arrowheads designate stamen AZs at the first position where intact cells were observed after organ removal.