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. 2017 Jan 6;17(1):4.
doi: 10.1186/s12870-016-0955-5.

Major Cys Protease Activities Are Not Essential for Senescence in Individually Darkened Arabidopsis Leaves

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Free PMC article

Major Cys Protease Activities Are Not Essential for Senescence in Individually Darkened Arabidopsis Leaves

Adriana Pružinská et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants.

Results: ABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves.

Conclusions: Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.

Keywords: Activity-based protein profiling; Papain-like proteases; Senescence; Vacuolar processing enzymes.

Figures

Fig. 1
Fig. 1
Individually darkened leaves on intact plants. a Five individually darkened leaves covered with aluminium foil. Arabidopsis plants (Col-0) were grown under short day conditions (8/16 hours day/night cycles). b Changes in chlorophyll ratio in individually darkened leaves. Each point represents the mean of means of 5 leaves from 4 individual plants with standard deviation (c) Changes in abundance of the large subunit of Rubisco (RBCL) in individually darkened leaves. d Expression of SAGs markers in individually darkened leaves: SAG12, SGR1 (Stay Green Gene 1), and PPase (Protein Phosphatase 2A Subunit A3, control)
Fig. 2
Fig. 2
Induced PLCP and reduced VPE activities individually darkened leaves. Increased DCG-04 (a) and MV151 (b) labeling of PLCPs during senescence. Leaf extracts of equal fresh weights of individually darkened leaves were labeled for 5 hours with 2 μM DCG-04 at pH 6.5 or 2 μM MV151 at pH 4.5 and biotinylated proteins were detected using streptavidin-HRP (a) or fluorescent proteins were detected by scanning (b), respectively. *, endogenously biotinylated protein. c, d Accumulation of RD21A (c) and AALP (d) proteins in individually darkened leaves. Protein extracts of equal fresh weights of individually darkened leaves were separated and detected from protein blots using RD21A and AALP –specific antibodies, respectively. e Reduced AMS101 labeling of VPEs in individually darkened leaves. Leaf extracts of equal fresh weights of individually darkened leaves were labeled for 2 hours with 2 μM AMS101 at pH5.5 and analysed by fluorescence scanning. Coomassie stains of the membranes (a-d) or protein gel (e) is used as a control to show degradation of the large subunit of Rubisco (RBCL). The dotted line (a, b) indicates a removed lane from a western blot
Fig. 3
Fig. 3
Extracts of senescent leaves contain more active PLCPs. a Profile of purified DCG-04-labelled and un-labelled proteins of control (day 0) and senescent leaves (day 7). Biotinylated proteins were purified from DCG-04 labelled proteomes using avidin beads. Four gel bands from control green leaves and four from yellow senescent leaves were excised and treated with trypsin. Eluted peptides were analysed and identified by MS/MS. b Spectral counts for identified PLCPs in the eight individual bands. c Sum of the total spectral counts over the 13 identified proteases, divided over green (day 0) and senescent (day 7) leaves
Fig. 4
Fig. 4
Major PLCP activities are depleted in senescent leaves of rd21A-1/aalp-1 double mutant plants without affecting Rubisco levels. a PLCP activity profiles of control (day 0) and senescent leaf (day 7) of rd21A-1, aalp-1, sag12-1 and ctb3-1 mutants and wild-type plants. b PLCP activity profiles of individually darkened leaves at 0, 3, 5 and 7 days of the rd21A-1/aalp-1 double mutant in comparison to wild-type (Col0) plants. Leaf extracts of equal fresh weights of individually darkened leaves were labeled for 5 hours with 0.2 μM DCG-04 at pH 6.5 and biotinylated proteins were detected from protein blots using streptavidin-HRP
Fig. 5
Fig. 5
Chlorophyll ratio is unaltered in individually darkened leaves for Cys protease mutant- and overexpressor lines. Chlorophyll ratio was measured with a SPAD meter on at least six leaves covered with aluminium foil for seven days from a total of eight plants. Error bars represent standard deviation of n = 8 biological replicates
Fig. 6
Fig. 6
Delayed onset of whole plant senescence in aalp-1 and rd21A-1/aalp-1 mutants. Plants were grown under long day conditions (16/8 day/night). Number of green (a) and yellow (/senescent) (b) leaves at different time points after the sowing of wild-type plants and aalp-1, rd21A-1/aalp-1 and rd21A-1 mutants. Each data point represents the mean of 16 plants with standard deviation from one representative experiment. The experiments were repeated twice with similar results

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