Senescence is induced in individually darkened Arabidopsis leaves, but inhibited in whole darkened plants

Abstract

It has long been known that leaf senescence can be induced in many plant species by detaching leaves and placing them in the darkness. It recently has been shown that entire Arabidopsis plants placed in the darkness are not induced to senesce, as judged by visible yellowing and certain molecular markers. Here, we show that when individual Arabidopsis leaves are darkened, but not when entire plants are darkened, senescence is induced in the covered leaves. This induction of senescence is highly localized. The phenomenon is leaf age dependent in that it occurs more rapidly and strongly in older leaves than in younger ones, as is the case with many forms of induced senescence. Whole adult plants placed in darkness, in contrast, show delayed senescence, although seedlings lacking primary leaves do not. These observations imply that the light status of the entire plant affects the senescence of individual leaves. A model summarizing the results is presented.

Figure 1

A, Time course of the whole-plant verses individual leaf darkness experiments. White bars indicate time in light and dark bars time in darkness. A synchronously growing population of Arabidopsis was established. Five days before the first harvest (day = −5), 5-d dark-treated plants were placed in the darkness or leaves were covered, whereas 2 d before harvest (day = −2), 2-d dark-treated plants were placed in the darkness or leaves were covered. Controls were harvested at the same time and were the same age as the dark treated plants. A second harvest was performed 3 d after the dark-treated plants/leaves had been returned to the light (d 3), to assay recovery. B, Photographs of representative leaves at the time of harvest from the whole-plant verse individual leaf darkness experiment diagrammed in A. C, Analysis of total chlorophyll and protein levels and gene expression (RNA or protein blots) in individually darkened leaves versus darkened plants, from the experiment diagrammed in A (d-0 samples). SAG12 is a known specific senescence-associated gene. BCB is a gene known to be induced by both senescence and darkness. Chlorophyll a/b-binding protein (CAB) is known to be associated with photosynthesis. 18S ribosomal RNA (rRNA) is used as a loading control for the RNA samples. “Older” leaves are leaf 5. “Younger” leaves are leaf 7. Chlorophyll and protein measurements were normalized separately for older and younger leaves such that the control value (undarkened leaves) was always 100. D, Analysis of total chlorophyll and protein levels in darkened leaves versus darkened plants before (d 0) and after (d 3) a return to the light, from the experiment diagrammed in A. Individual leaves or entire plants were darkened for the indicated times (treatment ended at d 0; controls were never darkened), and then returned to the light for 3 d (d 3), and total chlorophyll and protein levels determined as described in “Materials and Methods.” Results shown are for leaf 7. E, Petroleum jelly-covered leaves were not induced to senesce. Leaf 5 was covered with petroleum jelly, which induced no visible senescence after 5 d (untreated control shown inset; petroleum jelly-covered leaves are indicated by toothpicks). Three and 6 d later (8 d and 11 d covered), both the petroleum jelly-covered and control leaves went on to senesce at a similar rate. F, Leaves covered by black boxes, but not clear boxes, are induced to senesce. Leaf 5 was covered with shallow boxes made of black or clear x-ray film. Leaves covered with the black boxes were induced to yellow, whereas those covered with clear boxes were not.

Figure 2

Whole-plant darkness delays, but does not prevent, senescence. A, Time course of the experiment. White bars indicate time in light and dark bars time in darkness. A synchronously growing population of Arabidopsis was established, and when leaf 5 had begun to senesce but leaf 7 was still completely green, plants were transferred to the darkness (controls were left in the light). Plants were then harvested from both groups over a period of 6 d. B, Analysis of various parameters of senescence in “older leaves” (leaf 5). The upper graph shows both total chlorophyll levels (expressed as a percent of the level in the younger leaves at d 0; shown using bars), and chlorophyll a/b ratios (shown using lines). The lower graph shows total protein (also expressed as a percent of the level in the younger leaves at d 0). Blots shown are RNA or protein blots. SAG12 and SAG13 are both known senescence-associated genes, and SAG13 is known to be up-regulated during senescence earlier than SAG12. CAB is known to be associated with photosynthesis. C, As in B, but data shown are for “younger” leaves (leaf 7).

Figure 3

Darkness-induced promotion of senescence is very localized. Leaves were covered with mittens in which a hole had been punched (A). After 5 d, the darkened areas of the leaf had gone on to senesce, but the areas that received light did not (B).

Figure 4

Darkened seedlings. A, Time course of the experiments. Seedlings were grown on plates, and transferred to darkness and returned to the light as indicated. White segments indicate time in light and dark segments indicate time in darkness. Each bar represents a sample that was harvested at the indicated time. The first group of seedlings to be placed in the dark (a) had only cotyledons. The second group (b) had both cotyledons and primary leaves. B, Photographs of representative seedlings. The bottom row were all taken on d 19, when the “a” samples had been returned to light for 19 d and the “b” samples had been returned to light for 11 d. C, Total chlorophyll (expressed as a percent of d-0 controls) and chlorophyll a/b ratios of the seedlings.

Figure 5

hy2/hy3 double mutants and wild-type (Ler) plants respond similarly to both individual leaf and whole-plant darkness. Experiments were performed similarly to those in Figure 1 (d 0 samples), except that in this experiment the “older leaf” was leaf 4 and the “younger leaf” was leaf 5 (because in most instances the hy2/hy3 plants flowered after forming only five leaves). Whole plants or individual leaves were darkened for 5 d and then leaf 4 or 5 was excised from the plant for analysis. Controls were leaves 4 or 5 excised from age-matched, light-grown plants. Total chlorophyll levels were measured and normalized separately for the older and younger leaves and for each line such that the value of the control leaf was always 100%. Absolute chlorophyll levels of the hy2/hy3 plants were 80% of those of the Ler plants in the younger control leaf, and 63% of the Ler plants in the older control leaf. Relative total chlorophyll levels and chlorophyll a/b ratios are shown.

Figure 6

A model diagramming the promoting/delaying effects of darkness on senescence. The “default” effect of darkness (sensed locally, possibly at the level of the cell) appears to be to promote senescence. Age is a separate promoter of senescence, which is additive with darkness (and sensed at the level of the leaf). Superimposed over this, and epistatic to it, is the ability of darkness, when given at the level of the whole plant, to repress both age-mediated and individual leaf-mediated senescence (possibly due to a general inhibition of development). The inhibitory pathway is not present in detached leaves or cotyledons, however. NS, Non-senescent leaves; S, senescent leaves.