The RHA2a-interacting proteins ANAC019 and ANAC055 may play a dual role in regulating ABA response and jasmonate response

Abstract

Jasmonate- and ABA-mediated signalings are involved in the activation of defense responses of plants to biotic and abiotic stresses. Accumulating evidence has suggested the existence of comprehensive synergistic or antagonistic cross-talks between these two signaling pathways. However, relatively little is known about how these cross-talks are executed at the molecular level. Our recent works have implied that, ANAC019 and ANAC055, two highly related NAC family transcription factors in Arabidopsis, may play a dual role in regulating jasmonate response and ABA response.

We recently demonstrated that RHA2a, a C2H2-type RING finger protein, is an active E3 ligase and plays a positive role in regulating ABA-mediated control of seed germination and early seedling development.¹ In order to explore the substrates of RHA2a in ABA signaling, we conducted a yeast two-hybrid screen and identified ANAC019 and ANAC055, two highly related NAC family proteins,^2,3 as RHA2a interacting proteins. The physical interactions between RHA2a and the two NAC proteins were confirmed by in vitro pull-down assays.¹ The fact that RHA2a is an active E3 ligase prompted us to test the protein stability of ANAC019 and ANAC055. For these experiments, in vitro translated [³⁵S]ANAC019 and [³⁵S]ANAC055 polypeptides were incubated with wild-type Arabidopsis cellular extracts. In the absence of the proteasome specific inhibitor MG132, the levels of ANAC019 and ANAC055 decreased rapidly (Fig. 1A). But the presence of MG132 effectively inhibited the degradation of these in vitro translated proteins (Fig. 1B). As a control, an in vitro translated [³⁵S]Luciferase was found to be stable irrespective the presence or absence of MG132 (Fig. 1). The effect of MG132 on the stability of the two NAC proteins suggested that the protein abundance of ANAC019 and ANAC055 are likely regulated by the 26S proteasome-dependent proteolysis. Together, our results point to a possibility that the E3 ligase RHA2a might target ANAC019 and ANAC055 for degradation. To elucidate the physiological relevance of RHA2a interaction with the two unstable NAC proteins, we are rigorously investigating the possible role of ANAC019 and ANAC055 in ABA signaling during seed germination and early seedling development.

Figure 1.

Effect of MG132 on the stability of in vitro translated ANAC019 and ANAC055. (A) In vitro translated [³⁵S]ANAC019 and [³⁵S]ANAC055 were incubated with Col-0 cellular extracts for the indicated times in the absence of MG132. The incubation mixture were separated by 10% SDS-PAGE and analyzed by autoradiography. In vitro translated [³⁵S]Luciferase was used as control. The large subunit of the Rubisco complex (RbcL) stained with Coomassie blue was also shown as control. Arrow indicates the degraded protein ladder. (B) In vitro translated [³⁵S]ANAC019 and [³⁵S]ANAC055 proteins were incubated with Arabidopsis cellular extracts for 15 min in the absence (−) or presence (+) of MG132. The incubation mixture were separated by 10% SDS-PAGE and analyzed by autoradiography. In vitro translated [³⁵S]Luciferase was used as control. The large subunit of the Rubisco complex (RbcL) stained with Coomassie blue was also shown as a control. Arrow indicates the degraded protein ladder.

In another recent paper, we provided biochemical and genetic evidence showing that ANAC019 and ANAC055 function as transcription activators to regulate jasmonate-induced expression of defense genes.² The role of the two NAC genes in jasmonate signaling was examined with the anac019 anac055 double mutant and with transgenic plants overexpressing ANAC019 or ANAC055. The anac019 anac055 double mutant plants showed attenuated jasmonate-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) and LIPOXYGENASE2 (LOX2) expression, whereas transgenic plants overexpressing the two NAC genes showed enhanced jasmonate-induced VSP1 and LOX2 expression. That the jasmonate-induced expression of the two NAC genes depends on the function of COI1 and AtMYC2, together with the finding that overexpression of ANAC019 partially rescued the jasmonate-related phenotype of the atmyc2-2 mutant, led us to a hypothesis that the two NAC proteins act downstream of AtMYC2 to regulate jasmonate-signaled defense responses. Further evidence to substantiate this idea comes from the observation that the response of the anac019 anac055 double mutant to a necrotrophic fungus showed high similarity to that of the atmyc2-2 mutant.

The above-described findings raised a possibility that the NAC family proteins ANAC019 and ANAC055 play a dual role both in ABA signaling and jasmonate signaling. Consistent with this hypothesis we found that the ABA-induced expression of ANAC019 and ANAC055 were impaired in coi1-1,⁴ and atmyc2-2,^5,6 two jasmonate-related mutants (Fig. 2). Together with previous work showing that the coi1-1 and atmyc2-2 mutants also impairs jasmonate-induced expression of ANAC019 and ANAC055,² our results indicated that jasmonate—as well as ABA-induced activation of ANAC019 and ANAC055 expression requires the functions of the COI1 and AtMYC2 proteins, two of the known regulators of jasmonate-signaled processes in Arabidopsis, which suggests that ANAC019 and ANAC055 might be involved in the cross-talks between the jasmonate and ABA pathways.

Figure 2.

The coi1-1 and atmyc2-2 mutations affect jasmonate- and ABA-induced ANAC019 and ANAC055 expression. Two-week-old wild-type (Col-0) plants and coi1-1, atmyc2-2 mutants were treated with 50 µM MeJA (middle) or 50 µM ABA (right) and tissues were collected for RNA extraction at 6 h after treatment. Thirty micrograms of total RNA were loaded per lane, a duplicated gel stained with EtBr as loading control.

One possible explanation to this phenomenon came from the recent finding that ABA can induce the accumulation of jasmonate⁷ and, therefore, leads to the activation of the expression of the two NAC genes. Nevertheless, the work of Tran et al.⁸ provided strong evidence supporting an alternative explanation that ANAC019 and ANAC055 are involved in regulating drought tolerance of Arabidopsis plants. First, ANAC019 and ANAC055 bound specifically to a cis-element in the promoter region of ERD1, a dehydration responsive marker gene; Second, the expression of ANAC019 and ANAC055 was induced by dehydration, salt as well as the phytohormone ABA; Finally, transgenic plants overexpressing ANAC019 or ANAC055 showed increased expression of a group of stress-inducible genes and improved drought tolerance. These results suggested the possible involvement of ANAC019 and ANAC055 in regulating ABA-mediated drought tolerance of Arabidopsis plants. These, together with our work showing that ANAC019 binds to the promoter region of the VSP1 promoter raises the interesting question of how the two NAC transcription factors recognize the proper set of genes in response to a specific stress condition or hormonal stimulus. Using the yeast one-hybrid system, it has been determined the complete DNA binding sequence recognized by the NAC proteins, which contains the NAC recognition sequence CATGT and the core DNA binding site CACG.⁸ Our sequence examination in the VSP1 promoter region revealed cis-elements containing the exact NAC recognition site and core DNA bind site.² Furthermore, in vitro assay suggested ANAC019 physically bound to these elements.² It is reasonable to propose that the two NAC proteins might recognize the same elements to activate different sets of genes, other factors are therefore needed to guide the transcription factors to recognize the right set of genes in response to a specific stress condition.

Addendum to: Bu Q, Li H, Zhao Q, Jiang H, Zhai Q, Zhang J, Wu X, Sun J, Xie Q, Wang D, Li C. The Arabidopsis RING finger E3 ligase RHA2a is a novel positive regulator of ABA signaling during seed germination and early seedling development. Plant Physiol. 2009 doi: 10.1104/pp.109.135269. In press.

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Addendum to: Bu Q, Jiang H, Li CB, Zhai Q, Zhang J, Wu X, Sun J, Xie Q, Li C. Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses. Cell Res. 2008;18:756–767. doi: 10.1038/cr.2008.53.