Meagher Laboratory

Suppression of Reproductive ACTIN ACT1 Ectopic Expression Phenotypes by Co-expression of Reproductive PROFILIN 4 (PRF4) or ACTIN DEPOLYMERIZING FACTOR 7 (ADF7)

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms.  Plant genomes encode complex actin and actin-binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns.  In Arabidopsis, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture.  Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes.  We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs.  In order to examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically co-expressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1.  Our results demonstrated that the co-expression of these reproductive PRF4 (Figure 1) and ADF7 (Figure 2), but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture (Figure 3) to the double transgenic plants.  Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.  

 

 

 

Figure 1. Suppression of ACT1-induced dwarf phenotype by co-expression of pollen-specific PRF4.

(A) About 7-week-old plants.  WT, wild-type; A2P:A1, dwarf single transformant misexpressing ACT1; A2P:A1&P4, a double transformant misexpressing both ACT1 and PRF4 simultaneously.  Note the suppression of dwarf phenotype in the double transgenic plant.

(B) Rosette leaves of just bolted wild-type and single (A2P:A1) and double (A2P:A1&P4) transgenic plants. 

(C) Western blot analysis of ACT1 expression.  Top panel shows a blot probed with MAb45a for ACT1.  Both the dwarf single transformant misexpressing ACT1 and the normal double transgenic plant co-expressing ACT1 and PRF4 have same levels of ACT1 protein.  Wild-type has no detectable ACT1.  Bottom panel shows a Coomassie blue stained duplicate gel.

(D) Western blot analysis of PRF4 expression.  Top panel shows a blot probed with MAbPRF45 for PRF4 protein.  Only the double transgenic plant shows strong PRF4 band.  Bottom panel is a Coomassie blue stained duplicate gel to show equal loading of total protein.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Suppression of ACT1-induced dwarf phenotypeby co-expression of pollen/trichoblast-specific ADFs.

(A) - (C)ADF promoter-GUS fusion gene expression.

(A) Flower showing pollen-specific expression of ADF7.

(B) 2-d-old germinating seedling showing root hair-specific expression of ADF8.

(C) 2-week-old seedling showing constitutive expression of ADF9.

(D) 24-d-old wild-type (WT) plant, single transformants misexpressing ADF7 (A2P:ADF7) and ACT1 (A2P:A1) and a double transformant (A2P:A1&ADF7) co-expressing ACT1 and ADF7.

(E) Western blot analysis of ACT1 and ADF7 expression in leaves.  Top blot probed with reproductive actin-specific antibody MAb45a to show ACT1 protein.  The weak lower bands represent breakdown products. Middle blot from a duplicate gel is probed with MAbADF8, which recognizes ADF7.  Bottom blot is probed with anti-PEPC antibody to show equal loading of proteins.  Note the wild-type sample has no ACT1 and ADF7 proteins, the ADF7 misexpressing normal plant has no ACT1 protein, and the ACT1 misexpressing dwarf plant has no ADF7 protein. 

(F) 24-d-old wild-type (WT) plant, single transformants misexpressing ADF8 (A2P:ADF8) and ACT1 (A2P:A1) and a double transformant (A2P:A1&ADF8) co-expressing ACT1 and ADF8. (G) Western blot analysis of ACT1 and ADF8 expression in leaf samples. Top blot probed with MAb45a to show ACT1 protein.  The breakdown products were not detectable in this blot.  Middle blot from a duplicate gel is probed with MAbADF8 to show ADF8 protein.  Bottom panel probed with anti-PEPC antibody.

Figure 3. Immunofluorescence labeling of actin in leaf cells.

 

(A) and (B) Wild-type.

 

(C) and (D) Dwarf plant cells misexpressing ACT1.

 

(E) and (F) Cells from a suppressed, normal double transgenic plant co-expressing ACT1 and PRF4.

 

(G) Cells from a dwarf double transgenic plant co-expressing ACT1 and PRF1.

 

(H) Cells from a normal double transgenic plant co-expressing ACT1 and ADF7.

 

(I) Cells from a dwarf double transgenic plant co-expressing ACT1 and ADF9.

 

All samples were labeled with MAbGPa.  Bars = 20 µm.

 
 
   
Kandasamy et al., 2007