Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

Abstract

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.

Fig. 1.

Transcript accumulation in non-senescing (lane 1, no chlorotic symptoms) and senescing (lane 2; around 30% of the surface of each leaf showing chlorotic symptoms) rosette leaves of Arabidopsis thaliana plants. (A) KAT2 and KAT5 transcript levels were analysed by northern blot with 10 μg of total RNA samples. Ethidium bromide-stained ribosomal RNAs (rRNA) were included as loading control. (B) After hybridization with specific probes for KAT2 and KAT5 genes, the corresponding transcript levels were quantified by PhosphorImager analysis, the values normalized to the endogenous content of 18S ribosomal RNA and expressed as relative values to non-senescing leaves (mean values ±standard error from three replicate experiments). The CAB and SAG12 transcript levels were quantified by qRT-PCR. Values represent the mean ±standard error of three replicates and were normalized by the endogenous content of ACT2/8 transcript.

Fig. 2.

Dark-induced senescence in excised leaves of pKAT2::GUS transgenic plants. (A) The levels of KAT2 transcript were analysed by northern blot from total RNAs (10 μg) isolated from excised leaves of two independent pKAT2::GUS transgenic lines that were incubated in the dark for the indicated times in days after incubation in darkness. Ethidium bromide-stained ribosomal RNAs (rRNA) were included as loading control. (B) Quantification of KAT2 and SAG12 transcript levels by qRT-PCR at the indicated times in days after incubation in darkness. Values represent the mean ±standard error of three replicates and were normalized by the endogenous content of ACT2/8 transcript. (C) Excised leaves from both pKAT2::GUS transgenic lines were harvested at 0, 4, and 8 d after incubation in the dark, photographed, and then stained for GUS activity. Yellowing symptoms as a result of chlorophyll degradation correlated with GUS-stained leaves.

Fig. 3.

Luciferase-mediated luminescence in darkened pKAT2::LUC transgenic plants. Plants from two independent pKAT2::LUC transgenic lines, 1A in the top row and 12C in the bottom row as described in the scheme, were grown under short day photoperiodic conditions for 8 weeks and then moved to darkness. At the indicated times after the move plants were sprayed in the dark with 0.1 mM luciferin and the luminescence was monitored 4 h later by a low-light video CCD camera Hamamatsu C2400. The recorded luminescence images at the indicated times up to 10 d after the move to darkness are displayed in clock-wise sense. The colour code is an indication of luminescence intensity, blue being the lowest and red the highest level. The panel in the centre of the top row displays a clear field image of the plants before incubation in the dark.

Fig. 4.

Dark-induced senescence in excised leaves from wild-type and transgenic plants overexpressing KAT2 in sense (KAT2ox) or antisense (KAT2as) orientations as well as from the JA-insensitive coi1-1 mutant. (A) Plants from the indicated genotypes were grown under short day photoperiodic conditions for 6 weeks. Then, leaves were excised from the plants and incubated in the dark for 0, 4, and 8 d. At the indicated times leaves were photographed and subsequently processed for chlorophyll quantification. In the right panel, the figure shows 10-d-old seedlings of the different genotypes that were grown on MS-agar medium supplemented (+JA) or not (–JA) with 20 μM JA. (B) Quantification of chlorophyll a+b content of leaves at 0 d and 8 d after the move to darkness. Values are the mean of six independent replicates of 100 mg fresh weight of leaves. Error bars represent standard error. The experiment was repeated twice with similar results.

Fig. 5.

Kinetics of KAT2 and SAG12 transcript accumulation in dark-induced senescing excised leaves from wild-type Col and KAT2as transgenic plants. (A) The KAT2 (filled squares) and SAG12 (filled circles) transcript levels were quantified, by qRT-PCR as described before, in leaf samples excised from wild-type Col plants exposed to darkness for the indicated times (days in darkness; d.i.d). (B) SAG12 and KAT2 transcripts were quantified at 0 d.i.d (grey bars) and 8 d.i.d (black bars) in excised darkened leaves from wild-type Col and the KAT2as transgenic lines 16C and 12G. All values for transcript levels were normalized by the endogenous content of ACT2/8 transcript and expressed relative to the levels quantified for Col plants at day 0. The experiment was repeated three times with similar results.

Fig. 6.

Natural leaf senescence in wild-type and KAT2as transgenic plants. Every leaf from the rosette of wild-type Col and transgenic KAT2as lines 12G and 16C plants grown under long day photoperiodic conditions for 8 weeks were excised and the chlorophyll (a+b) contents quantified and plotted versus leaf position. Values are the mean of four independent replicate samples (three leaves per sample) and the error bars corresponded to the standard error. Values are expressed relative to the chlorophyll content of the youngest leaf in each genotype.