HD-ZIP III activity is modulated by competitive inhibitors via a feedback loop in Arabidopsis shoot apical meristem development

Abstract

Shoot apical meristem (SAM) development is coordinately regulated by two interdependent signaling events: one maintaining stem cell identity and the other governing the initiation of lateral organs from the flanks of the SAM. The signaling networks involved in this process are interconnected and are regulated by multiple molecular mechanisms. Class III homeodomain-leucine zipper (HD-ZIP III) proteins are the most extensively studied transcription factors involved in this regulation. However, how different signals are integrated to maintain stem cell identity and to pattern lateral organ polarity remains unclear. Here, we demonstrated that a small ZIP protein, ZPR3, and its functionally redundant homolog, ZPR4, negatively regulate the HD-ZIP III activity in SAM development. ZPR3 directly interacts with PHABULOSA (PHB) and other HD-ZIP III proteins via the ZIP motifs and forms nonfunctional heterodimers. Accordingly, a double mutant, zpr3-2 zpr4-2, exhibits an altered SAM activity with abnormal stem cell maintenance. However, the mutant displays normal patterning of leaf polarity. In addition, we show that PHB positively regulates ZPR3 expression. We therefore propose that HD-ZIP III activity in regulating SAM development is modulated by, among other things, a feedback loop involving the competitive inhibitors ZPR3 and ZPR4.

Figure 1.

Disrupted SAM and Partially Abaxialized Leaves in zpr3-1d. (A) Phenotypes of zpr3-1d and zpr3-2 compared with the wild type (Col-0). A full-size ZPR3 cDNA was overexpressed under the control of the CaMV 35S promoter to confirm the zpr3-1d phenotype (35S:ZPR3). Five-week-old plants were photographed. (B) Partial abaxialization of the zpr3-1d leaves. The third rosette leaves harvested 10 d after stratification were examined by scanning electron microscopy. Note that the adaxial epidermal cells of the mutant have irregular shapes like the abaxial epidermal cells of the wild type. Bar = 50 μm. (C) Upregulation of some genes promoting abaxial cell identity in zpr3-1d. Transcript levels were measured by real-time RT-PCR using the aerial parts of 10-d-old plants grown on Murashige and Skoog (MS)-agar plates. Means + se are shown (n = 3). (D) Transcript levels of the HD-ZIP III genes in zpr3-1d. Transcript levels were measured by real-time RT-PCR using the aerial parts of 10-d-old plants grown on MS-agar plates. Means + se are shown (n = 3). (E) Absence of secondary shoots in zpr3-1d. Inset shows an enlarged view of a zpr3-1d cauline leaf axil. Note bare axil, indicative of no lateral meristem (arrow). (F) Schematic of the T-DNA insertion sites in the genomes of zpr3-1d (white triangle) and zpr3-2 (black triangle). Chr 3, chromosome 3. (G) Phenotypes of the 35S:ZPR3 transgenic plants. Two major types of the apices observed in 10-d-old seedlings are shown: one with a pin-like structure and the other without a SAM (left panel), longitudinal sections of which are shown in the right top and bottom panels, respectively. (H) Transgenic plants overexpressing ZPR3 under the control of an estradiol-inducible promoter. Only newly emerging leaves were curled downward after estradiol induction (20 μM) 22 d after stratification (bottom panel). Transcript levels were measured by RT-PCR. A tubulin gene (TUB) was used as control for constitutive expression.

Figure 2.

Interactions of ZPR3 with HD-ZIP III Proteins via the ZIP Motifs. (A) In vitro pull-down assays. A recombinant GST-ZPR3 fusion or GST alone was incubated with in vitro–translated, radiolabeled HD-ZIP III proteins. Bound HD-ZIP III proteins were separated on a 12% SDS-PAGE and subjected to autoradiography. (B) Mapping of the interacting domain in PHB. The numbers denote residue positions for each construct (schematics are shown in the left panel). Interactions were examined both by growth of yeast cells in selective media (-QD) and by in vitro pull-down assays (right panel). IN, input; Y2H, yeast two-hybrid assays; HD, homeodomain; ZIP, leucine zipper; START, StAR-related lipid transfer; AA, amino acids. (C) Interactions of ZPR3 with PHB and REV. Total cellular extracts from N. benthamiana cells expressing MYC fusions of PHB (PH), REV (RE), and ATHB2 (HB) were incubated with GST or GST-ZPR3. Protein gel blots of interacting proteins probed with an anti-MYC antibody are shown. The amounts of the MYC fusion proteins were immunologically normalized using the same antibody before use (Input). Coomassie blue–stained gels are displayed at the bottom to show the amounts of GST and GST-ZPR3 used. (D) Subcellular localization of ZPR3. A ZPR3-GFP fusion was transiently expressed in onion epidermal cells.

Figure 3.

Inhibition of Dimerization and Activity of PHB by ZPR3. (A) PHB dimerization assays by yeast coexpression. ZPR3 was expressed under the control of the Met-suppressible promoter (pMET25) (MET25:ZPR3). The ZPR3 gene is not expressed on selective media without Leu, Trp, and His (-LWH) but is expressed on selective media without Leu, Trp, His, and Met (-LWHM). Cell growth on the -LW media indicates that the yeast cells contain the two plasmid DNAs. The ΔC construct of PHB includes residues 1 to 174. (B) PHB dimerization and activity assays by β-galactosidase (β-Gal) activity measurements. For the measurements of activities in transcription-activation, a BD-PHB fusion construct containing a full-size PHB protein was used. The β-Gal activities were normalized by dividing total activity by optical cell density. Means + se are shown (n = 3). (C) Effects of ZPR3 on PHB protein stability. An anti-MYC antibody was used to immunologically measure the levels of the MYC-PHB proteins in transgenic plants coexpressing a MYC-PHB fusion under the CaMV 35S promoter and ZPR3 under the estradiol-inducible promoter. The Coomassie blue–stained gel is displayed at the bottom. (D) Effects of ZPR3 on PHB transcription. Transcript levels in the plants from (C) were measured by RT-PCR. A tubulin gene (TUB) was used as control for constitutive expression. (E) A genetic cross between phb-1d-Col and zpr3-1d. The inset shows an enlarged view of the phb-1d-Col plant. Four-week-old plants were photographed. (F) Comparison of the inflorescences of the parental mutants and the zpr3-1d phb-1d-Col double mutant. ZPR3 transcript levels were measured by RT-PCR (bottom panel). A tubulin gene (TUB) was used as control for constitutive expression. (G) Comparison of flowers and leaves of the parental mutants and the zpr3-1d phb-1d-Col double mutant. The fifth leaves were photographed. (H) A genetic cross between zpr3-1d and transgenic plants overexpressing REV (35S:REV). Three-week-old plants were photographed.

Figure 4.

Phenotypes of zpr3-2 and zpr3-2 zpr4-2. (A) T-DNA insertion site (arrow) in the zpr3-2 genome. The black box denotes a translating exon, and the white boxes denote nontranslating exons. Transcript levels were measured by RT-PCR (right panel). A tubulin gene (TUB) was used as control for constitutive expression. (B) Phenotype of an adult zpr3-2 mutant compared with the wild type. Four-week-old plants were photographed. (C) Inflorescences of zpr3-2 compared with the wild type. An enlarged view of a mutant node with three inflorescences (IN) is shown in the bottom panel. (D) Short internodes of zpr3-2. Distribution of wild-type and mutant internode lengths. (E) Phenotype of zpr3-2 zpr4-2 compared with the wild type at seedling (top) and adult (bottom) stages. The images were obtained by scanning electron microscopy. The mutant seedling has three cotyledons (top panel) 9 d after stratification. Note the clustering of cauline leaves due to short internodes (bottom panel). Bars =1 mm. (F) Phenotype of zpr3-2 zpr4-2 compared with the wild type 27 d after stratification. Left top panel, control plant; the other three panels, the mutant plants. (G) Phenotype of zpr3-2 zpr4-2 compared with the wild type 45 d after stratification. (H) Phenotypic comparison of wild-type, phb-1d, and zpr3-2 zpr4-2 plants. Five-week-old plants were photographed.

Figure 5.

Altered SAM Morphologies and Flower and Leaf Phenotypes in zpr3-2 zpr4-2. (A) SAM morphology of zpr3-2 zpr4-2. The left top panel shows the SAM of a wild-type control plant. The other panels show the SAM of the double mutant at different growth stages. Arrows indicate the developing leaf primordia. DAS, days after stratification. Bars = 50 μm. (B) A vertical section of zpr3-2 zpr4-2 apex. Arrows indicate the leaf primordia that are ectopically produced. The SAM-like structure is observed at the center of this section. Bar = 50 μm. (C) Various types of zpr3-2 zpr4-2 SAMs 18 d after stratification. Bars = 100 μm. (D) Abnormal floral structure of zpr3-2 zpr4-2 compared with the wild type. In some flowers, multiple carpels were formed. (E) Ovule-like appearance on the surface of zpr3-2 zpr4-2 ovary (arrow). (F) Scanning electron micrograph showing papillae-like structures at the petal margin in zpr3-2 zpr4-2. These characteristic are indicative of homeotic transformation. Bar = 200 μm. (G) Scanning electron micrograph of the inflorescence stem of zpr3-2 zpr4-2. It is fasciated (top panel), and ectopic leaves are produced on the surface (bottom panel). Bars = 500 μm.

Figure 6.

Phenotype of phv Compared with the Wild Type and zpr3-2 zpr4-2. Note that the leaf number of the triple mutant is similar to that of the wild type control plant. Five-week-old plants were photographed.

Figure 7.

Altered Expression Patterns of SAM-Related Genes in zpr3-2 zpr4-2. (A) Expression of CLVs and WUS in zpr3-2 zpr4-2 compared with the wild type. Transcript levels were measured by real-time RT-PCR. Shoot apex tissues of 2-week-old plants were used for total RNA extractions. Means + se are shown (n = 3). (B) In situ hybridization showing the expression domains of CLV3 in the zpr3-2 zpr4-2 apex compared with the wild type. The apex region was sectioned and probed either with a sense (negative control) or an antisense CLV3 probe. Bars = 50 μm. (C) Expression of CLVs and WUS in phb-1d compared with the wild type. Transcript levels were measured by real-time RT-PCR. Shoot apex tissues of 2-week-old plants were used for total RNA extractions. Means + se are shown (n = 3).

Figure 8.

A Schematic Model for ZPR3 Function. ZPR3 negatively regulates HD-ZIP IIIs at the protein level. However, it is positively regulated by the HD-ZIP III genes at the transcriptional level, forming a negative feedback loop. miR165/166 does not directly interact with ZPR3 but regulates SAM development via the HD-ZIP IIIs.