ORE9, an F-box protein that regulates leaf senescence in Arabidopsis

Abstract

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.

Figure 1.

Age-Dependent Senescence Symptoms in the ore9-1 Mutation. (A) The age-dependent senescence phenotype of wild-type (Columbia [Col]) and mutant leaves. Photographs show representative leaves at each time point. (B) Survival curve. The graph shows the percentage of leaves alive on a given DAE. n = 100. (C) to (E) Chlorophyll content (C), photochemical efficiency of PSII (D), and membrane ion leakage (E) were examined every 4 days from 12 DAE, when the fourth rosette leaves were just fully grown. Fv/Fm, maximum quantum yield of PSII electron transport (maximum variable fluorescence/maximum yield of fluorescence). Error bars indicate sd; n = 48. (F) Age-dependent changes of gene expression. Total cellular RNA was extracted at 12, 20, and 24 DAE from wild-type leaves and at 12, 20, 24, 28, 32, and 36 DAE from ore9-1 leaves.

Figure 2.

Delay of Leaf Senescence in the ore9-1 Mutant during Senescence Accelerated by Plant Hormones. The change of chlorophyll content and photochemical efficiency in continuous light (A), ABA-induced senescence (B), MeJA-induced senescence (C), and ethylene-induced senescence (D). (E) shows the change in expression of the SEN4 gene. Fv/Fm, maximum quantum yield of PSII electron transport (maximum variable fluorescence/maximum yield of fluorescence). Error bars indicate sd; n = 24. Total cellular RNA was extracted from control (C) and hormone-treated (T) leaves.

Figure 3.

Map-Based Cloning of ORE9. The numbers of recombination events between the CAPS markers and the ORE9 locus (r) are shown. The hatched bar represents the 4.5-kb fragment used for a complementation experiment. Ch. 2, chromosome 2; BAC, bacterial artificial chromosome; cM, centimorgan; MAP3K, mitogen-activated protein kinase kinase kinase.

Figure 4.

Molecular Analysis of the ORE9 Gene. (A) Predicted amino acid sequence of the ORE9 gene. The F-box and the LRRs are noted by a black box and by underlining, respectively. The mutated residue in the ore9-1 mutation is indicated by an asterisk. (B) Scheme of the predicted structures of the ORE9 and ore9 proteins. The F-box and the LRRs are noted by the letter F and by white boxes, respectively. (C) Alignment of the F-box motifs from various proteins and an F-box consensus sequence generated from an alignment of 38 F-box–containing proteins (Patton et al., 1998). Highly conserved residues are capitalized. (D) Alignment of the 18 LRRs in ORE9. Highly conserved residues are boxed in black; gray shading indicates functionally conservative substitutions in more than half of the comparisons.

Figure 5.

Interaction of ORE9 and the Arabidopsis Homolog of Yeast Skp1, ASK1, in a Yeast Two-Hybrid Experiment. (A) Scheme of the ORE9 and ASK1 constructs used in the yeast two-hybrid analysis. Solid and hatched boxes represent the GAL4 binding domain (BD) and the GAL4 activation domain (AD), respectively. The F-box and the LRRs are noted by the letter F and by white boxes, respectively. (B) Plasmid pairs used in the yeast two-hybrid experiment. (C) and (D) Yeast cells transformed with the plasmid pairs were cultured either on control synthetic minimal medium containing 2% dextrose but lacking tryptophan and leucine (C) or on viability test medium (synthetic minimal medium lacking tryptophan, leucine, and histidine and containing 2 mM 3-amino-1,2,4-triazole) (D). (E) β-Galactosidase activities of the transformants.

Figure 6.

Interaction of ORE9 and ASK1 in an in Vitro Binding Assay. (A) The ORE9 and ASK1 constructs used in the in vitro binding analysis. The cross-hatched box represents the GST protein. The F-box and the LRRs are noted by the letter F and by white boxes, respectively. (B) In vitro binding experiment for analysis of the ORE9-ASK1 interaction. The in vitro translation products of ORE9(1-693), ORE9(1-326), and ORE9(56-693) are shown in lanes 1, 2, and 3, respectively. The ³⁵S-labeled proteins were incubated with resin-bound GST (lanes 4, 5, and 6) or GST-ASK1 (lanes 7, 8, and 9).