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. 2011 Mar;23(3):1000-13.
doi: 10.1105/tpc.111.083089. Epub 2011 Mar 29.

The Jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect Jasmonate-regulated stamen development in Arabidopsis

Susheng Song  1 , Tiancong Qi, Huang Huang, Qingcuo Ren, Dewei Wu, Changqing Chang, Wen Peng, Yule Liu, Jinrong Peng, Daoxin Xie
Affiliations

The Jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect Jasmonate-regulated stamen development in Arabidopsis

Susheng Song et al. Plant Cell. 2011 Mar.

Abstract

The Arabidopsis thaliana F-box protein CORONATINE INSENSITIVE1 (COI1) perceives jasmonate (JA) signals and subsequently targets the Jasmonate-ZIM domain proteins (JAZs) for degradation by the SCF(COI1)-26S proteasome pathway to mediate various jasmonate-regulated processes, including fertility, root growth, anthocyanin accumulation, senescence, and defense. In this study, we screened JAZ-interacting proteins from an Arabidopsis cDNA library in the yeast two-hybrid system. MYB21 and MYB24, two R2R3-MYB transcription factors, were found to interact with JAZ1, JAZ8, and JAZ11 in yeast and in planta. Genetic and physiological experiments showed that the myb21 myb24 double mutant exhibited defects specifically in pollen maturation, anther dehiscence, and filament elongation leading to male sterility. Transgenic expression of MYB21 in the coi1-1 mutant was able to rescue male fertility partially but unable to recover JA-regulated root growth inhibition, anthocyanin accumulation, and plant defense. These results demonstrate that the R2R3-MYB transcription factors MYB21 and MYB24 function as direct targets of JAZs to regulate male fertility specifically. We speculate that JAZs interact with MYB21 and MYB24 to attenuate their transcriptional function; upon perception of JA signal, COI1 recruits JAZs to the SCF(COI1) complex for ubiquitination and degradation through the 26S proteasome; MYB21 and MYB24 are then released to activate expression of various genes essential for JA-regulated anther development and filament elongation.

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Figures

Figure 1.
Figure 1.
Interactions of JAZs with MYB21 and MYB24 in the Y2H System. (A) Y2H assay to detect interactions of JAZs with MYB21 and MYB24. Twelve Arabidopsis JAZs were individually fused with the LexA DNA binding domain (BD) in pLexA. MYB21 and MYB24 were individually fused with the activation domain (AD) in pB42AD. Interactions of JAZs with the AD domain in the pB42AD empty vector were used as negative controls. Interactions (represented by blue color) were assessed on 2% Gal/1% raffinose/SD/-Ura/-His/-Trp/-Leu/X-β-Gal medium. (B) Immunoblot analysis of JAZ proteins expressed in yeast strains from (A). Total proteins were extracted from the yeast strains of (A) and analyzed by immunoblot using anti-LexA antibody. Expression of the JAZ proteins was detected as expected (Chung and Howe, 2009).
Figure 2.
Figure 2.
MYB21 and MYB24 Interact with JAZ1, JAZ8, and JAZ11 in N. benthamiana. (A) to (D) LCI assays show that Arabidopsis MYB21 and MYB24 interact with Arabidopsis JAZ8 and JAZ11 in N. benthamiana. MYB21 and MYB24 were fused with N-terminal fragment of LUC (nLUC) to generate MYB21-nLUC and MYB24-nLUC, respectively. JAZ8 and JAZ11 were fused with the C-terminal fragment of LUC (cLUC) to produce cLUC-JAZ8 and cLUC-JAZ11, respectively. The leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the indicated construct pairs. The data were collected 50 h after infiltration. (E) BiFC assay indicates that Arabidopsis MYB21 and MYB24 interact with Arabidopsis JAZ1 in N. benthamiana. MYB21 and MYB24 were fused with the C-terminal fragment of yellow fluorescence protein (cYFP) to form cYFP-MYB21 and cYFP-MYB24. JAZ1 was fused with N-terminal fragment of YFP (nYFP) to form JAZ1-nYFP. YFP fluorescence was detected in N. benthamiana leaves coinfiltrated with combinations of JAZ1-nYFP/ cYFP-MYB21, and JAZ1-nYFP/ cYFP-MYB24. The positions of nuclei were shown by 4′,6-diamidino-2-phenylindole (DAPI) staining.
Figure 3.
Figure 3.
JAZ8 and JAZ11 Interact with the N Terminus of MYB21 and MYB24. (A) Schematic diagram of MYB21 and MYB24 domain constructs. MYB21 and MYB24 were separated into two parts: the MYB21NT/MYB24NT part containing conserved R2 (red) R3 (blue) domain, and the MYB21CT/MYB24CT part containing the NYWG/SM/VDDI/LWS/P motif (green). MYB21NT/MYB24NT and MYB21CT/MYB24CT were ligated into pB42AD vector for fusion with the AD domain individually. (B) Y2H assays show that JAZ8 and JAZ11 interact with MYB21NT and MYB24NT in yeast. The interactions were observed on 2% Gal/1% raffinose/SD/-Ura/-His/-Trp/-Leu/X-β-Gal medium. (C) LCI assays show that JAZ8 interacts with MYB24NT in N. benthamiana. MYB24NT and MYB24CT shown in (A) were fused with nLUC to produce MYB24NT-nLUC and MYB24CT-nLUC, respectively. JAZ8 was fused with cLUC to produce cLUC-JAZ8. Signal was detected in N. benthamiana leaves 50 h after coinfiltration with the construct pairs indicated in the white circle.
Figure 4.
Figure 4.
The Jas Domain in the C-Terminal Part of JAZ Proteins Is Required for Interaction with MYB21 and MYB24. (A) Schematic diagram of JAZ8 domain constructs. The diagram shows the conserved ZIM (black) and Jas (gray) domains. Different domains of JAZ8 were fused with the BD domain. (B) Y2H assay for interactions of JAZ8 domain constructs with MYB21 and MYB24. MYB21 and MYB24 were fused with the AD domain individually. (C) Schematic diagram of JAZ11 domain constructs. There are two ZIM domains (black) and two Jas domains (gray) in JAZ11. (D) Y2H assay for interactions of JAZ11 domain constructs with MYB21 and MYB24. [See online article for color version of this figure.]
Figure 5.
Figure 5.
Transgenic Expression of MYB21 Partially Rescues the Male Fertility of coi1-1. (A) Comparison of anthers and flowers in different genotypes as indicated. The flower in coi1-1 MYB21OE1 shows dehisced anther (top panel) and elongated filaments (bottom panel) compared with coi1-1. Pollen grains from coi1-1 fail to germinate in vitro (middle), whereas transgenic expression of MYB21 rescues the pollen germination of coi1-1 (right middle). (B) The ratio of filament length to pistil length in Columbia-0 (Col-0), coi1-1, and coi1-1 MYB21OE1. Error bars represent se (n = 10). (C) Quantitative real-time PCR analysis of MYB21 transcription level in young flower buds from Col-0, coi1-1, and coi1-1 MYB21OE1 using ACTIN8 as the internal control. Error bars represent se. (D) Comparison of seed set in different genotypes as indicated. (E) Seed numbers per silique in Col-0, coi1-1, and coi1-1 MYB21OE1. For coi1-1 MYB21OE1, the seed numbers per silique were calculated from 10 randomly selected fertile siliques. Error bars represent se (n = 10). (F) Main inflorescences in Col-0, coi1-1, and coi1-1 MYB21OE1. Asterisks indicate fertile siliques in coi1-1 MYB21OE1.
Figure 6.
Figure 6.
MYB21 Is Not Required for JA-Regulated Root Growth Inhibition, Anthocyanin Accumulation, and Defense against Insect Attack. (A) Relative root length of 9-d-old seedlings grown on MS medium containing indicated concentrations of MeJA. Relative root length is shown as a percentage of root length on MS medium. Error bars represent se (n = 20). (B) Anthocyanin contents of seedlings grown on MS medium containing indicated concentrations of MeJA. Error bars represent se. FW, fresh weight. (C) Top panel shows the phenotype of 35-d-old seedlings grown in a B. impatiens–infested growth chamber. Bottom panel shows young B. impatiens from indicated soil pots. (D) Plant survival rates of the indicated plants after incubation with B. impatiens at the indicated time points. [See online article for color version of this figure.]
Figure 7.
Figure 7.
Excess Expression of MYB21 Causes Male Sterility. (A) Flowers of Col-0 and two MYB21 overexpression lines (MYB21OE4 and MYB21OE5). (B) Main inflorescences. Arrows indicate sterile siliques in MYB21OE4. (C) Quantitative real-time PCR analysis of the MYB21 expression level in young flower buds of the indicated plants using ACTIN8 as the internal control. Error bars represent se.
Figure 8.
Figure 8.
Excess Expression of MYB24 Causes Male Sterility. Quantitative real-time PCR analysis (top panel) of the MYB24 expression level in young flower buds of Col-0 and six MYB24 transgenic lines as indicated (bottom panel). ACTIN8 was used as the internal control. Error bars represent se. Asterisks in the bottom panel indicate fertile siliques in MYB24 transgenic lines. [See online article for color version of this figure.]
Figure 9.
Figure 9.
Excess Expression of MYB21 Fails to Rescue Male Fertility in coi1-1. (A) Flowers of Col-0, coi1-1, and three MYB21 transgenic lines in the coi1-1 background as indicated. (B) Main inflorescences of (A). Asterisks indicate fertile siliques in coi1-1 MYB21OE1. (C) Quantitative real-time PCR analysis of the MYB21 expression level in young flower buds. Seven male-sterile coi1-1 plants transgenic for MYB21 contain >8-fold wild-type level of MYB21, coi1-1MYB21OE2, and coi1-1MYB21OE26 are shown as representatives. ACTIN8 was used as the internal control. Error bars represent se.
Figure 10.
Figure 10.
Homomeric and Heteromeric Interactions of MYB21 and MYB24. (A) to (C) LCI assays show the interactions of MYB21-MYB21 (A), MYB24-MYB24 (B), and MYB21-MYB24 (C) in N. benthamiana leaves. Leaf regions were coinfiltrated with Agrobacterium strains containing the indicated constructs. The data were collected 50 h after infiltration. (D) Y2H assays show interactions among MYB21NT, MYB24NT, MYB21, and MYB24 in yeast. The interactions were observed on 2% Gal/1% raffinose/SD/-Ura/-His/-Trp/-Leu/X-β-Gal medium.
Figure 11.
Figure 11.
A Simplified Model for the Molecular Mechanism of JA-Regulated Male Fertility. The Jas domain of JAZ proteins (JAZ) interact with the R2R3 domain of MYB21 and MYB24 (MYB) to attenuate transcriptional function of MYB21 and MYB24 and inhibit expression of downstream genes (Early genes) leading to male sterility; upon perception of the JA signal, COI1 recruits JAZs to the SCFCOI1 complex for ubiquitination and degradation through the 26S proteasome. MYB21 and MYB24 (MYB) are then released to activate expression of downstream genes essential for JA-regulated stamen development. [See online article for color version of this figure.]
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