Senescence-associated gene expression in ozone-stressed Arabidopsis leaves
Open Access
- Author:
- Miller, Jennifer Dawn
- Graduate Program:
- Plant Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 14, 2000
- Committee Members:
- Eva Joy Pell, Committee Chair/Co-Chair
Richard N Arteca, Committee Member
Ramesh Raina, Committee Member
Allen T Phillips, Committee Member - Keywords:
- gene expression
Arabidopsis thaliana
ethylene
ozone
senescence - Abstract:
- The similarities and differences between natural leaf senescence and O3-induced accelerated leaf senescence were assessed by comparing the expression levels of senescence-associated genes (SAGs) and photosynthesis-associated genes (PAGs) in Arabidopsis thaliana. During natural leaf senescence SAG transcript levels increase while PAG transcript levels decline. SAG and PAG expression levels were determined by northern analysis in Arabidopsis ecotype Landsberg erecta plants treated with 0.15 mL L-1 O3 for 6 hours per day for 14 days. Ozone treatment caused the early expression of BCB, ERD1, SAG13, SAG18, SAG20, SAG21 and CCH and an early decline in rbcS and cab transcript levels. No induction of the following senescence-related genes was found: SAG12, SAG19, MT1 and Atgsr2. The spatial distribution of SAG expression in leaves was investigated by treating transgenic plants carrying the SAG13 promoter-GUS construct with O3. SAG13 promoter-driven GUS activity was located throughout O3-treated leaves. This expression pattern differed from that found in naturally senescing leaves. While differences do exist between natural leaf senescence and O3-induced accelerated leaf senescence, the process in O3-treated leaves is highly regulated and involves the expression of many genes associated with natural senescence. Protein levels for two SAGs were investigated during O3 treatment to determine whether the increases in SAG transcript levels were accompanied by corresponding increases in protein levels. ERD1 and BCB protein levels were determined by western analysis in plants treated with 0.15 mL L-1 O3 for 6 hours per day for 8 and 14 days. ERD1 transcript levels were strongly induced by O3 treatment, whereas ERD1 protein levels declined with increasing O3 exposure. BCB transcript levels also increased during O3 treatment, while no BCB protein accumulation was detected during the exposure. The reason for the disparity between transcript and protein levels is not known, causing the functional significance of O3-induced increases in ERD1 and BCB transcript levels to be questioned. The plant hormone ethylene modulates the timing of leaf senescence and is produced during exposures to high doses of O3, so the role of ethylene in inducing SAG expression during O3 treatment was studied. SAG and PAG transcript levels were measured by northern analysis in wild-type Arabidopsis, ecotype Columbia, plants and the ethylene-insensitive mutant, etr1-3. During exposure to 0.15 mL L-1 O3 for 6 hours per day for 14 days, equivalent changes in SAG and PAG expression levels were found in wild-type and etr1-3 plants. The increase in SAG transcripts and decline in PAG transcripts occurred without any increase in ethylene production, ACC levels or ACS6 transcript levels. During exposure to a higher O3 concentration, 0.35 mL L-1 O3 for 6 hours, ethylene production was induced, but once again the level of SAG induction and PAG transcript decline were similar in wild-type plants and etr1-3 mutants. This work suggested that ethylene perception was not necessary for the induction of SAG transcripts and decline in PAG transcripts during O3 exposure. The etr1-3 mutant retains some partial sensitivity to ethylene, so the role of ethylene in O3-induced SAG expression was investigated in three additional ethylene-insensitive mutants. Wild-type plants and ethylene-insensitive mutants etr1-3, ein2-1, ein3-1 and ein4-1 were treated with 100 mL L-1 ethylene for 6 hours and expression of the ethylene-responsive gene, HEL, was measured by northern analysis to determine the ethylene sensitivity of leaves. Transcript for HEL was induced in ethylene-treated wild-type plants and some induction was also found in ethylene-insensitive mutants, etr1-3 and ein3-1. No ethylene induction of HEL was found in ein2-1 or ein4-1 mutants. The ethylene-insensitive mutants were treated with O3 to see if SAGs could be induced in the absence of ethylene perception, as in ein2-1 and ein4-1 leaves, or under reduced ethylene perception, as in etr1-3 and ein3-1 leaves. Wild-type plants and etr1-3, ein2-1, ein3-1 and ein4-1 mutants were treated with 0.15 mL L-1 O3 for 3.5 hours and SAG transcript levels were determined. SAGs were induced in wild-type plants and all ethylene-insensitive mutants at similar levels. Wild-type plants and mutants were treated with 0.30 mL L-1 O3 for 3.5 hours to determine SAG expression levels when the O3 treatment caused a sharp increase in ethylene production. In the presence of O3-induced ethylene production, SAGs were once again induced at similar levels in wild-type plants and ethylene-insensitive mutants. This work demonstrates that ethylene is not the primary signal for SAG expression during O3 treatment.