AtEXP2 is involved in seed germination and abiotic stress response in Arabidopsis

Abstract

Expansins are cell wall proteins that promote cell wall loosening by inducing pH-dependent cell wall extension and stress relaxation. Expansins are required in a series of physiological developmental processes in higher plants such as seed germination. Here we identified an Arabidopsis expansin gene AtEXPA2 that is exclusively expressed in germinating seeds and the mutant shows delayed germination, suggesting that AtEXP2 is involved in controlling seed germination. Exogenous GA application increased the expression level of AtEXP2 during seed germination, while ABA application had no effect on AtEXP2 expression. Furthermore, the analysis of DELLA mutants show that RGL1, RGL2, RGA, GAI are all involved in repressing AtEXP2 expression, and RGL1 plays the most dominant role in controlling AtEXP2 expression. In stress response, exp2 mutant shows higher sensitivity than wild type in seed germination, while overexpression lines of AtEXP2 are less sensitive to salt stress and osmotic stress, exhibiting enhanced tolerance to stress treatment. Collectively, our results suggest that AtEXP2 is involved in the GA-mediated seed germination and confers salt stress and osmotic stress tolerance in Arabidopsis.

Figure 1. Expression pattern of AtEXP2 gene.

(A) Tissue-specific expression of AtEXP2. Various tissues of Arabidopsis wild type plants were harvested for RNA extraction. (B) Time course of AtEXP2 expression. Dry seeds and imbibed seeds of Col-0 were harvested for RNA extraction. Transcript levels of AtEXP2 were measured by real-time RT-PCR, and the values were normalized against the levels of TUB2 as a control. Error bars represent SD. (C) GUS staining in germinating seeds of the pAtEXP2:GUS transgenic line. Seeds from T₃ homozygous plants of the pAtEXP2:GUS transgenic line were analyzed. Bar = 1 mm.

Figure 2. Relative expression levels of AtEXP2 in exp2 and 35S:AtEXP2 line.

Seeds of wild type, exp2 and 35S:AtEXP2 overexpression line were harvested for RNA extraction after 24 h imbibition in water. Transcript abundance was measured by real-time RT-PCR and the values were normalized against the levels of TUB2 as a housekeeping gene. Error bars represent SD.

Figure 3. Germination phenotype of the wild type, exp2 and 35S:AtEXP2 line.

Non-dormant seeds of wild type, exp2 and 35S:AtEXP2 overexpression line were employed in the germination assay. The germination frequencies were scored daily until the 7th day after sown. Error bars represent SD. A Student’s t-test was calculated at the probability of either 5% (*P<0.05) or 1% (**P<0.01).

Figure 4. Expression of AtEXP2 in response to exogenous GA application.

Col-0 wild type seeds were harvested 4 h, 6 h and 8 h after imbibition in 10 µM GA₃ solution for RNA extraction respectively. Transcript levels were measured by real-time RT-PCR, and the values were normalized against the levels of TUB2 as a control. Error bars represent SD. A Student’s t-test was calculated at the probability of 1% (**P<0.01).

Figure 5. Expression of AtEXP2 in response to paclobutrazol treatment.

AtEXP2 expression was determined by quantitative real-time RT-PCR in 24 h imbibed seeds treated with 10 µM PAC or without (Mock). Error bars represent SD. A Student’s t-test was calculated at the probability of 5% (*P<0.05).

Figure 6. Germination phenotype of the wild type, exp2 and 35S:AtEXP2 line in response to GA and PAC treatment.

Seeds of wild type, exp2 and 35S:AtEXP2 line were treated with 10 µM GA₃ (A), 1 µM (B) or 5 µM (C) paclobutrazol (PAC). Error bars represent SD. A Student’s t-test was calculated at the probability of either 5% (*P<0.05) or 1% (**P<0.01).

Figure 7. Effects of DELLA on AtEXP2 expression during seed germination.

Expression level of AtEXP2 was measured in 24 h imbibed seeds of wild type, ga1-3, and various DELLA mutants. penta indicates the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant. Error bars represent SD. A Student’s t-test was calculated at the probability of 1% (**P<0.01).

Figure 8. Germination phenotype of the wild type, exp2, and 35S:AtEXP2 line in response to abiotic stresses.

Seeds of wild type, exp2 and 35S:AtEXP2 line were treated with different concentrations of NaCl (100 or 200 mM), sucrose (150 or 250 mM) and mannitol (200 or 400 mM). Error bars represent SD. A Student’s t-test was calculated at the probability of either 5% (*P<0.05) or 1% (**P<0.01).

Figure 9. Expression of AtEXP2 in response to salt and osmotic stresses during seed germination.

Col-0 seeds were collected 24 h after imbibition in different concentrations of NaCl, sucrose and mannitol. Error bars represent SD. A Student’s t-test was calculated at the probability of 1% (**P<0.01).