The Non-JAZ TIFY Protein TIFY8 of Arabidopsis thaliana Interacts with the HD-ZIP III Transcription Factor REVOLUTA and Regulates Leaf Senescence

Abstract

The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53. As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV's function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.

Keywords: Arabidopsis thaliana; JAZ proteins; PEAPOD; REVOLUTA; TIFY8; jasmonic acid signaling; leaf senescence; transcription factor regulation.

Figure 1

Yeast two-hybrid interactions between REV and TIFY8 and truncated proteins. (a) Scheme of TIFY8 and the truncated versions used in the yeast two-hybrid assay. The yellow box represents the TIFY/ZIM domain. TIFY8 full length (1 to 361aa), TIFY8-N (1 to 229aa), TIFY8-N1 (1 to 176aa), TIFY8-C (176 to 361aa), TIFY8-C1 (230 to 361aa), TIFY8-C2 (284 to 361aa), TIFY8-C3 (176 to 229aa), and TIFY8-C4 (239 to 283aa). (b) Representative yeast two-hybrid assay between GAL4-BD-REV and TIFY8, as well as a series of truncated versions of the TIFY8 protein shown in (a), fused with GAL4-AD. A serial 1:10 dilution of each transformed yeast was spotted onto control (DDO) and different protein–protein interaction selective media with increasing stringency. Blue boxes indicate interactions, and light blue boxes indicate weak interactions.

Figure 2

In planta protein–protein interaction between TIFY8 or TIFY8-truncated versions (see Figure 1a) with REV using BiFC in transiently transformed Arabidopsis protoplasts or tobacco leaves. Protoplasts were analyzed with the cytoflex cell sorter (left), orange squares indicate transformed protoplasts (RFP), and purple squares indicate interaction via BiFC (YFP). Transiently transformed Nicotiana benthamiana leaves were analyzed under the confocal laser scanning microscope (right). YFP indicates BiFC; RFP is used as a transformation control. Scale bar indicates 20 µm.

Figure 3

Arabidopsis protoplasts were transiently transformed with a 2.8-kbp-fragment of the WRKY53 promoter fused to the GUS gene as a reporter construct; 35S: REVd and 35S: TIFY8 constructs were used as effector plasmids. These transfected protoplasts were simultaneously incubated with 40 µM JA or the same volume of water for the MOCK condition. A boxplot of the values relative to the empty vector control is presented (n = 7). One-way ANOVA test was performed, lowercase letters indicate significant differences among groups (p ≤ 0.05).

Figure 4

Boxplots presenting the expression of REVOLUTA, WRKY53, and TIFY8 over time in leaf tissue of Arabidopsis wildtype plants Col-0. Leaves No. 6 and 7 of each rosette were harvested from 4- to 7-week-old plants. Expression levels were determined via qRT-PCR, and values were normalized to ACTIN2 (n = 3). In the case of TIFY8, the ratio for the two existing splicing variants (SV1, SV2, see Figure S2) was determined.

Figure 5

Photosynthetic parameters analyzed for the senescence phenotyping of tify8-4 and TIFY8-OE compared to rev5 mutants and wildtype Col-0 plants. (a) The Automated Colorimetric Assay (ACA) to categorize the color of individual leaves of at least six plants pixelwise into five categories: green, green-yellow, yellow, brown, and purple. The percentage of each group with respect to total pixel number of all leaves (1–10) is presented (n = 6). (b) Boxplot of Fv/Fm values measured with PAM for leaves No. 5 of 4- to 7-week-old plants (n = 6–8). (c) Boxplot of the decrease in solute retention determined through ion leakage in leaves No. 4 of 4- to 7-week-old plants (n = 6–8). One-way ANOVA test was performed, lowercase letters indicate significant differences among groups (p ≤ 0.05). (d) Gene expression of the senescence-associated marker genes SAG12 was analyzed by qRT-PCR and normalized to the expression of the ACTIN2 gene (mean values ± SD, n = 3).

Figure 6

Boxplot of JA concentrations measured by LCMS in Col-0, rev5, tify8-1T, and tify8-4 mutants and TIFY8-OE plants. The concentration was determined in pools of leaves No. 5 to 9 of 4- to 7-week-old-plants (n = 4).

Figure 7

Heat map of the gene expression measured by qRT-PCR of REV, TIFY8, and WRKY53 in Col-0, rev5, and tify8 mutant plants for 3-week-old and 5-week-old plants 6 h, 24 h, and 96 h after JA treatment. Expression is shown as log2-fold changes relative to the respective MOCK treatments; blue color indicates repression, whereas red color indicates induction.

Figure 8

Yeast two-hybrid interactions between PEAPODs, PPD2-truncated proteins, TIFY8, and REV. (a) Scheme of the PPD1, PPD2, TIFY8, and truncated PPD2 versions used in the yeast two-hybrid assay. The orange box represents the PEAPOD domain (PPD), the yellow box represents the TIFY/ZIM domain, and the green box represents the Jas-like domain (Jas*). PPD2 full length (1 to 318aa), PPD2-N1 (1 to 117aa), PPD2-N (1 to 204aa), PPD2-C1 (117 to 204aa), PPD2-C (117-316aa), and PPD2-C2 (205 to 316aa). (b) Representative yeast two-hybrid assay between GAL4-BD-TIFY8 or GAL4-REV and PPDs, as well as a series of truncated versions of the PPD2 protein shown in (a) fused with GAL4-AD. A serial 1:10 dilution of each transformed yeast was spotted onto control (DDO) and different protein-protein interaction selective media with increasing stringency. Blue boxes indicate interactions.

Figure 9

Yeast two-hybrid interactions between REV and JAZ proteins. Representative yeast two-hybrid assay between Gal4-BD-REV and GAL4-AD-JAZ fusion proteins. A serial 1:10 dilution of each transformed yeast was spotted onto control (DDO) and different protein–protein interaction-selective media with increasing stringency. Blue boxes indicate interactions. Protein domains of the different JAZ proteins are indicated on the right. The yellow box represents the TIFY/ZIM domain. The green box represents the Jas domain. The red box indicates the repressing EAR domain.

Figure 10

Model of the development-specific regulatory effects of TIFY8, PPDs, and JAZs on REV. (1) TIFY8, which is highly expressed during early development, blocks WRKY53 expression by its interaction with REV. How REV activates other early developmental genes is still unclear and needs most likely additional factors or modifications. In this stage, JAZ as well as PPD expression is low. (2) In juvenile leaves, JAZs and PPDs are expressed, but JA levels are still low; therefore, even enhanced repression of WRKY53 is achieved by the additional complex formation of JAZs/PPDs with REV. (3) At the transition from maturation to senescence, JA levels increase, and JA-Ile mark the JAZs and PPDs for degradation via the 26S proteasome through the interaction with the F-box protein COI1. However, TIFY8 has no Jas domain and cannot be marked for degradation by JA. Instead, expression of the TIFY8 gene is strongly reduced. Both the JA-dependent and the JA-independent pathways lead to an activation of gene expression of REV target genes, including WRKY53, which then activates other WRKYs, other TFs, and additional SAGs. TF: transcription factors; SAGs: senescence-associated genes.