Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity

Abstract

Auxin regulates a variety of physiological and developmental processes in plants. Although auxin acts as a suppressor of leaf senescence, its exact role in this respect has not been clearly defined, aside from circumstantial evidence. It was found here that ARF2 functions in the auxin-mediated control of Arabidopsis leaf longevity, as discovered by screening EMS mutant pools for a delayed leaf senescence phenotype. Two allelic mutations, ore14-1 and 14-2, caused a highly significant delay in all senescence parameters examined, including chlorophyll content, the photochemical efficiency of photosystem II, membrane ion leakage, and the expression of senescence-associated genes. A delay of senescence symptoms was also observed under various senescence-accelerating conditions, where detached leaves were treated with darkness, phytohormones, or oxidative stress. These results indicate that the gene defined by these mutations might be a key regulatory genetic component controlling functional leaf senescence. Map-based cloning of ORE14 revealed that it encodes ARF2, a member of the auxin response factor (ARF) protein family, which modulates early auxin-induced gene expression in plants. The ore14/arf2 mutation also conferred an increased sensitivity to exogenous auxin in hypocotyl growth inhibition, thereby demonstrating that ARF2 is a repressor of auxin signalling. Therefore, the ore14/arf2 lesion appears to cause reduced repression of auxin signalling with increased auxin sensitivity, leading to delayed senescence. Altogether, our data suggest that ARF2 positively regulates leaf senescence in Arabidopsis.

Fig. 1.

Whole-plant phenotypes of Arabidopsis wild-type (‘Col’, left) and ore14-1 mutant (right) plants at 35, 46, and 65 d after germination (DAG).

Fig. 2.

ORE14 is auxin response factor 2 (ARF2). (A) Map-based cloning of ORE14. The number of recombination events between CAPS markers and the ORE14 locus (r) is shown; BAC, bacterial artificial chromosome; cM, centimorgan. (B) Schematic representation of ORE14 with positions of the ore14-1 and 14-2 mutations; B3, DNA binding domain; ARF, auxin response region; AUX/IAA, domain involved in dimerization with other ARFs or Aux/IAA.

Fig. 3.

Extended leaf longevity in ore14/arf2 mutants. (A) Age-dependent senescence phenotype of the third and fourth rosette leaves of wild-type (‘Col’) and ore14-1/arf2-10 mutant plants at different ages. The photographs show representative leaves at each time point. (B) Survival curve. Values indicate the percentage of leaves alive on a given day after emergence (DAE). n=100. (C–E) Chlorophyll content (C), photochemical efficiency (F_v/F_m) of PSII (D), and membrane ion leakage (E) were measured from the third and fourth leaves starting at 12 DAE, when leaves had just reached full growth. Error bars indicate standard deviation (SD, n=30). (F) Age-dependent changes in gene expression. Total cellular RNAs isolated at the indicated ages were probed with CAB, RBCS, SEN4, and SAG12.

Fig. 4.

Delayed leaf senescence of ore14/arf2 mutants during dark-induced senescence. (A) Phenotypes of wild-type and ore14 mutant leaves. The third and fourth rosette leaves were detached at the age of 12 d and incubated in darkness; DAT, days after treatment. (B–D) Chlorophyll content (B), photochemical efficiency (F_v/F_m) of PSII (C), and membrane ion leakage (D) were examined every 2 d after dark treatment. Error bars indicate SD (n=24). (E) Expression of SEN4. Total cellular RNAs were isolated from wild-type and ore14-1/arf2-10 mutant leaves at the days indicated.

Fig. 5.

Delay of leaf senescence in the ore14-1/arf2-10 mutant during senescence accelerated by phytohormones. The third and fourth rosette leaves were detached at the age of 12 d and incubated under continuous light in MES buffer alone (A), with 50 μM ACC (B), 100 μM MJ (C), or 50 μM ABA (D). Chlorophyll content and photochemical efficiency are presented as average percent values ±SD, relative to those of leaves incubated in light; n=24. DAT, days after treatment.

Fig. 6.

Delay of leaf senescence in the ore14-1 mutant during senescence accelerated by oxidative stress. The third and fourth rosette leaves were detached at the age of 12 d and incubated under continuous light in MES buffer alone (A) and with 15 mM hydrogen peroxide (B). Chlorophyll content and photochemical efficiency are presented as average per cent values ±SD, relative to those of leaves incubated in light (n=24). DAT, days after treatment.

Fig. 7.

Hypocotyl growth inhibition responses to exogenous phytohormones. Seedlings of wild type (‘Col’) and the ore14-1 mutant were grown for 4 d in various concentrations of NAA (A), ACC (B), or BA (C). The auxin resistant 1-3 (axr1-3) mutant was included as an internal control. At least 20 seedlings were sampled for each treatment. Bars indicate standard errors.