Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid

Abstract

Abscisic acid (ABA) mediates plant responses to environmental stress, particularly to water status. During germination, the embryo emerges from dormancy as the ABA concentration declines. Exposure to exogenous ABA during germination arrests development rapidly, but reversibly, enabling seedlings to withstand early water stress without loss of viability. Postgermination proteolytic degradation of the essential ABI5 transcription factor is interrupted by perception of an increase in ABA concentration, leading to ABI5 accumulation and reactivation of embryonic genes. Making use of the ABA-hypersensitive hyl1 mutant of Arabidopsis, we show that the ABA signal is transmitted to the transcriptional apparatus through mitogen-activated protein kinase signaling.

Fig 1.

ABI5 protein and mRNA accumulation in wild-type and hyl1 seedlings. Wild-type and hyl1 seeds were stratified at 4°C for 3 days with or without 0.7 μM ABA and then transferred to constant light at 22°C. Seeds were harvested at the indicated times after transfer and total protein and RNA were isolated. (A) Western blot analysis using antibodies to ABI5. Each lane contained 10 μg protein (asterisk marks an empty lane caused by sample loss). (B) Northern blot analysis. Each lane contained 10 μg total RNA. (C) RNA was isolated from germinating seeds of transgenic plants containing a 35S-ABI5 cDNA construct, hyl1 seeds, and wild-type seeds 3 days after stratification. Each lane contained 10 μg total RNA.

Fig 2.

Kinase assays using MBP and ABI5 substrates. (A) Wild-type and hyl1 seedlings were germinated in liquid medium for 6 days. ABA (in 0.1 mM NaOH) was added at the indicated concentrations (nothing was added to the control, C) and the seedlings were harvested after 15 min. Protein extracts (30 μg) were fractionated on a 10% polyacrylamide gel containing MBP as a kinase substrate, renatured, and the gels were incubated with ³²P-ATP. (B) Quantification of PhosphorImager band intensities for the 42- and 46-kDa MAPK activities detected in A. (C) Wild-type and hyl1 seedlings were germinated as in A; 10 μM ABA (in 0.1 mM NaOH) was added to the medium, and seedlings were harvested at the indicated times. Protein extracts were prepared and used for an in-gel kinase assay as described in A, but with ABI5 as substrate. (D) Wild-type and hyl1 seedlings were germinated as in A; 10 μM ABA (in 0.1 mM NaOH) was added to the medium, and seedlings were harvested at the indicated times; proteins were extracted and analyzed as described in A with MBP as kinase substrate. (E) Wild-type seeds and seeds from transgenic plants expressing a 35S-AtMPK3 cDNA or a 35S-AtMPK3 dsRNA construct were germinated as in A, then either 0.1 mM NaOH (−) or 20 μM ABA in 0.1 mM NaOH (+) was added 30 min before harvesting. Proteins were fractionated and assayed for MAPK activity as in A. (F) Leaves of Nicotiana benthamiana seedlings grown at 22°C were infiltrated with an Agrobacterium strain carrying a CaMV 35S promoter fusion expressing an HA epitope-tagged AtMPK3. After 4 days, the leaves received the following treatments: C, none; S, 0.07% EtOH for 5 min; ABA, 50 μM ABA in 0.07% EtOH for 5 min; H₂O, water for 15 min; and H₂O₂, 20 mM for H₂O₂ for 15 min. The infiltrated leaves were then harvested and proteins were extracted. HA-tagged AtMPK3 was immunoprecipitated with anti-HA antibodies, incubated with ³²P-ATP and MPB, then fractionated on a 15% polyacrylamide gel.

Fig 3.

Relative amounts of MAPK and other gene transcripts in wild-type and hyl1 plants. Wild-type and hyl1 seeds were plated on MS medium with (+) or without (−) 0.7 μM ABA. The seeds were harvested and used for RNA isolation 3 days after stratification. Equal amounts of RNA from each sample were used for RT-PCR analysis. The sequences of primers used to detect expression of the indicated genes in A and B are given in Materials and Methods. Gene abbreviations: ABI1, -3, and -5 are the genes identified by the abi1, -3, and -5 mutations and encode a protein phosphatase and two transcription factors, respectively; AtMPK1–9 are the genes that encode A. thaliana MAP kinases 1–9; the ANP1 gene encodes a MAPKKK that activates the stress kinase cascade. Ubiquitin mRNA served as a reference. (C) RT-PCR analysis of AtMPK3 and ubiquitin mRNA levels in wild-type (wt) and transgenic A. thaliana plants expressing a CaMV 35S promoter-driven inverted repeat AtMPK3 cDNA construct (dsRNA) or a 35S-AtMPK3 cDNA construct (35S-MPK3).

Fig 4.

Transgenic plants overexpressing MPK3 are hypersensitive to ABA. Seeds of wild-type, hyl1, and two independent 35S-AtMPK3 cDNA transgenic plants (AtMPK3–1 and –2) were stratified at 4°C for 2 days and then transferred to constant light at 22°C. Pictures were taken 10 days (0 and 0.6 μM ABA) and 20 days (3 μM) after stratification.

Fig 5.

Kinase inhibitors decrease sensitivity to ABA-mediated postgermination arrest. Wild-type and hyl1 seeds were plated on filter paper saturated with MS solution alone or MS containing either PD98059 (100 μM) or K252a (1 μM). ABA was present at the indicated concentrations. After 2 days at 4°C, seeds were transferred to constant light at 22°C. Pictures were taken 10 days after stratification.