Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases

Abstract

The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.

Figure 1

Homology to ABI1 among Arabidopsis PP2Cs. The 40 most closely-related proteins to ABI1 (BLASTP) were aligned by the Pileup program of GCG using progressive pairwise alignments and the phylogenetic tree was constructed by the GCG GrowTree program. Capital letters on right correspond to clades classified by Schweighofer et al. (2004).

Figure 2

Hierarchical clustering meta-analysis (Zimmerman et al. 2005) of Affymetrix 22k ATH1 gene expression profiles (Schmid et al. 2005) of the 28 Arabidopsis PP2C gene family members homologous to ABI1. Capital letters spanning genes in the cladogram correspond to clades classified by Schweighofer et al. (2004) shown in Fig. 1. Results are given as heat maps in gray scale coding (absolute signal values, darker = higher expression). (A) Growth stages of the life cycle, starting with mature siliques (seeds). (B) Plant tissue- and organ-specific expression. Growth stage annotations are based on the Boyes key (2001). The choice of organ terminology is based on vocabulary defined by the Plant Ontology Consortium (http://www.plantontology.org).

Figure 2

Hierarchical clustering meta-analysis (Zimmerman et al. 2005) of Affymetrix 22k ATH1 gene expression profiles (Schmid et al. 2005) of the 28 Arabidopsis PP2C gene family members homologous to ABI1. Capital letters spanning genes in the cladogram correspond to clades classified by Schweighofer et al. (2004) shown in Fig. 1. Results are given as heat maps in gray scale coding (absolute signal values, darker = higher expression). (A) Growth stages of the life cycle, starting with mature siliques (seeds). (B) Plant tissue- and organ-specific expression. Growth stage annotations are based on the Boyes key (2001). The choice of organ terminology is based on vocabulary defined by the Plant Ontology Consortium (http://www.plantontology.org).

Figure 3

Functional analysis of Arabidopsis ABI1-Like PP2Cs in transient gene expression assays. The y-axis has been normalized by a scalar applied to all data to reflect fold-induction relative to control (no ABA = 1.0) so that different experimental datasets (A- C) are more easily compared. Overexpression of ABI2, HAB2, AP2C7, AP2C15, (A), or AP2C9 and AP2C18 (B) cDNAs have antagonist effects, whereas overexpressed AP2C1 has agonist (inductive) effects (C) on ABA-inducible ProEm:GUS expression in separate maize mesophyll protoplast experiments. Number with symbol “x” represents fold transactivation by AP2C1 relative to ABA-treated control. Negative numbers next to bars indicate the percent inhibition of ProEm:GUS expression relative to controls. The abi1^null and abi1-1 effector constructs were included as negative and positive controls, respectively. Control samples were co-transformed with a Pro35S-ppdk:ABI1^null expression construct encoding the phosphatase inactive G174D mutant (Sheen, 1998). The AP2C1m construct contains the analogous (G→ D178) point mutation as abi1-1 dominant negative (G→ D180) allele. Data are the average of at least two replicates, ± SE.

Figure 4

Cre-lox recombination-ready acceptor vector for transient plant expression assays. pCR701: loxH alone. pCR702- 705 plasmids contain 2xHIS6, 2x-HA, 2x-FLAG, and 2x-cMYC epitope tags, respectively (see Table S2), upstream (5’) and in frame with loxH recombination site to give N-terminal fusion peptides with acceptor pUNI cDNA plasmids. Cis-acting element symbols are not shown to scale.

Figure 5

Immunoblots of protein extracts from maize protoplasts transformed with standard constructs (ProUbi:HA∷abi1-1, ProUbi:LUC). (A) Increasing amounts of transformed DNA for ProUbi:LUC reporter or (B) ProUbi:HA∷abi1-1 effector show that transgene activities are correlated with protein expression. The amounts of electroporated DNAs and their relative protein activities in the same samples are shown under the lanes of the immunoblots. The relative inhibition of ABA-inducible gene expression by abi1-1 protein (45 kD) was measured by ProEm:GUS/ProUbi:LUC enzyme assays as shown in Fig. 3.

Figure 6

Functional and expression analyses of select ABI1-Like PP2Cs using the Cre-lox acceptor vectors pCR701, pCR703 and pCR704. (A) Transient expression of ProUbi:FLAG∷AP2C2 and ProUbi:HA∷AP2C7 effects on ABA-inducible ProEm:GUS reporter gene expression. Asterisk (*) indicates significantly different than control (P < 0.0008, two sided Student’s t-test, equal variance assumed. The positive control VP1 and ABF3 statistical treatments are not shown to emphasize the test). The y-axis has been normalized by a scalar applied to all data to reflect fold-induction relative to control (no ABA = 1.0). Data are means of four replicates, ± SE. (B- D) Immunoblots of protein extracts from samples for panel A. Five μg ProUbi:LUC and 20 μg ProUbi:FLAG∷AP2C2 or mock control (NoDNA) were co-electroporated into protoplasts and cultured overnight. Fifteen μg protein extract was run on SDS-PAGE, transferred to PVDF membrane, and probed with anti-FLAG antibody (B, left side), then stripped and re-probed with anti-LUC antibody (B, right side). M= protein standard marker. (C) Immunoblots probed for ProUbi:HA∷AP2C2 (left) and ProUbi:cMYC∷AP2C2 (right). The predicted recombinant AP2C2 protein is 46 kD. (D) Immunoblots probed for ProUbi:FLAG∷AP2C8 and FLAG∷AP2C2 (left; proteins are predicted 46 kD) and ProUbi:HA∷AP2C7 and HA∷PP2CA (right; predicted proteins are 55 and 49 kD, respectively).