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Materials and Methods
- Senescence Is Induced in Individually Darkened Arabidopsis Leaves, but Inhibited in Whole Darkened Plants

Plant Materials and Treatments

Seeds of Arabidopsis ecotype Landsberg erecta were originally obtained from the Arabidopsis Stock Center at Ohio State University (Columbus). Plants were grown on Fafard germination mix (Conrad Fafard Inc., Agawam, MA) under continuous cool-white fluorescent light (120 µmol m2 s-1) in growth chambers at 22°C. Under these conditions, plants flowered after forming approximately eight rosette leaves (cotyledons were not counted). Because the age of a leaf can affect its response to factors that influence senescence, plants were taken from synchronously growing populations and, with the exception of the seedling experiments, only identically aged leaves were pooled (e.g. the fifth and seventh true leaves were pooled separately). In most experiments, leaf 5 was used as an "older" leaf (beginning to visibly yellow in controls at the time of harvest) and leaf 7 was used as a "younger" leaf (fully expanded but showing no visible signs of senescence in controls; see Fig. 1B). Plants at this stage had flowered and contained developing siliques. This stage corresponds to d 0 in both Figure 1A (in which the experiment was then nearly finished) and Figure 2A (in which the experiment was then just beginning). At d -5 in Figure 1A, plants had bolted but had few or no siliques.

For the whole-plant dark treatment, whole, soil-grown plants in their pots were placed in dark boxes in the same chambers in which they had initially been grown. For the individual leaf dark treatments, leaves were covered with cloth "mittens" as shown in Figure 3 (in most experiments the mittens did not have holes in them, however).

In the seedling experiments, plants were grown in culture on agar-solidified medium containing 0.65 g L-1 Peters Excel 15-5-15 fertilizer (Grace Sierra, Milpitas, CA).

RNA Extraction and Blotting

Total RNA was extracted using RNA Isolator (Genosys Biotechnologies, The Woodlands, TX). RNA was size fractionated by electrophoresis on 1% (v/v) formaldehyde-agarose gels and transferred onto nylon membranes by capillary blotting. Fifteen micrograms of total RNA was loaded in each lane. Probes were 32P-labeled by random priming (Prime-a-Gene kit, Promega Corporation, Madison, WI). Hybridization was done at 65°C overnight in 0.25 M NaH2PO4 (pH 7.4), 7% (w/v) SDS, 1% (w/v) casein, and 1 mM EDTA, and membranes were washed two times for 45 min each in 0.04 M NaH2PO4 (pH 7.2), 1% (w/v) SDS, and 1 mM EDTA. Probe hybridization was visualized with a phosphorimager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


Leaf extracts were prepared by grinding tissue under liquid N2 and adding equal volumes of lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% [v/v] NP40, 0.1% [w/v] SDS, 0.1% [v/v] Triton X-100, 0.7% [v/v] 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride). Samples were vortexed, centrifuged, and the supernatant added to an equal volume of 2× load buffer (125 mM Tris-HCl, pH 7.5, 1% [v/v] 2-mercaptoethanol, 4% [w/v] SDS, 20% [v/v] glycerol, and 0.01% [w/v] bromphenol blue). Equal volumes of each sample (representing protein derived from equal volumes of leaf tissue) were electrophoresed on SDS-PAGE gels and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Immunodetection was performed as by Shanklin et al. (1987). The CAB antibody is described by Sigrist and Staehelin (1992), and recognizes the LHC2b family of proteins.

Total Protein and Chlorophyll Extraction and Quantitation

Single Arabidopsis leaf discs were frozen in liquid nitrogen, ground in 2.2 mL of 96% (v/v) ethanol, incubated at room temperature in the dark for 30 min, pelleted in a microcentrifuge, and the chlorophyll content of the supernatant quantified spectrophotometrically using the method of Wintermans and DeMots (1965). The pellet was then rinsed once with 96% (v/v) ethanol, allowed to air dry, and resuspended in 60 µL of 1% (w/v) SDS, 1% (v/v) NP40, and 25 mM Tris, pH 7.5, by vortexing and heating for 30' at 70°C. Protein was then quantified using the Bio-Rad DC Protein Assay kit according to the manufacturer's instructions (Bio-Rad Laboratories; protein for immunoblots was extracted differently, however, as described above). For each experimental condition, protein and chlorophyll concentrations were determined for three to eight independent leaf discs (from three-eight separate leaves) and the results averaged. Normalization was performed as described in the figure legends. Error bars show 1 SD. In the seedling experiments, seedlings were pooled and chlorophyll concentrations determined from an aliquot of the pooled tissue. Three replicate measurements were made for each pool and the results averaged.

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