One plant actin isovariant, ACT7, is induced by auxin and required for normal callus formation

Abstract

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.

Figure 1.

Reactivity of Anti-Actin Antibodies. (A) Phylogenetic relationship of the eight expressed actins of Arabidopsis (left) and the specificity of monoclonal antibodies (right). Veg, vegetative; Rep, reproductive; MAb, monoclonal antibody. (B) Protein gel blot analysis showing differential binding of the antibodies with the eight Arabidopsis recombinant actins (3 μg/lane) and actins in pollen and seedling extracts (25 μg total protein/lane). (C) Immunofluorescence staining of actin filaments in Arabidopsis Fi-3 and tobacco BY-2 suspension cells. Note that all of the antibodies except MAb45a detected dense arrays of actin filaments in both cell types. Bar = 25 μm.

Figure 2.

Histochemical GUS Staining of Control and Callus-Induced Roots. Roots from Arabidopsis transgenic plants harboring four different actin gene promoter–GUS fusions were incubated on CIM containing 1 mg/L 2,4-D and 50 μg/L kinetin for 7 days (7d) or 21 days (21d) and then stained for GUS expression. Note that the strongest staining of root-derived callus tissue is from the ACT7-GUS transformant. Portions of callus from the ACT1-GUS transformant also show strong staining. Control roots were grown on germination medium without hormones. Bar = 2 mm.

Figure 3.

Auxin Rapidly Accelerates ACT7-GUS Fusion Gene Expression in Transgenic Arabidopsis. Wild-type and transgenic root samples were incubated on germination medium supplemented with or without (control) 2,4-D for 1 hr and then analyzed for GUS gene expression. Small root fragments were incubated in 4-methylumbelliferyl-β-d-glucuronide substrate, and GUS activity was assayed at different time intervals to validate the linearity of the response. GUS activity is measured in arbitrary fluorescence units (see Methods) per 2-mg sample. An average of four independent readings is presented with the standard error for each sample.

Figure 4.

Differential Expression of Actin Isovariants during Hormone-Induced Callus Formation in Wild-Type Arabidopsis. (A) Protein gel blot analysis of actin from seedlings grown in quarter-strength liquid germination medium with (3d and 8d) or without (Con) 2,4-D treatment. Identical blots containing 25 μg of total protein per lane were probed with the general antibody MAbGPa and the subclass-specific antibodies MAb13a, MAb45a, and MAb2345a. The numbers below the blots indicate the relative quantities of actin, with 1.0 being the highest amount in each blot. Similar results were obtained with IAA treatment (not shown). (B) Protein gel blot analysis of actins in leaves after a 2-week incubation on solid medium with (CIM) and without (germination medium [GM]) hormones. (C) Protein gel blot analysis of actins from root explants after 7 and 21 days of treatment (7d and 21d) on CIM. ACT1 and ACT11 recombinant proteins were used as controls to show the reactivity of the antibodies.

Figure 5.

ACT1 Is Expressed Only in a Subset of Vegetative Tissues. (A) to (F) Histochemical GUS staining of a leaf (A), hypocotyl (B), and roots ([C] to [F]) of transgenic Arabidopsis plants containing ACT1-GUS fusions. (C) and (D) show a root tip (C) and a portion of root showing a lateral root primordium (D) before hormone treatment. (E) and (F) show roots incubated for 7 days on hormone-containing CIM. (G) to (I) Confocal images of hormone-treated root callus tissue (21 days) double labeled with the general polyclonal anti-actin antibody PAbGPa and the reproductive actin-specific monoclonal antibody MAb45a. Actin filaments stained with PAbGPa are shown in green (G), and those stained with MAb45a are shown in red (H). The filaments appear yellow where the green and red signals overlap, as shown in (I). (J) to (L) Immunofluorescence staining of 3-month-old root-derived callus tissue. Note that MAbGPa (J) and MAb2345a (K) stain actin filaments in all cells, whereas MAb45a (L) stains actin filaments only in a single cell in the field shown. Bars in (A) and (E) = 500 μm; bar in (B) = 250 μm; bars in (C) and (F) = 100 μm; bar in (D) = 50 μm; bars in (G), (J), (K), and (L) = 25 μm.

Figure 6.

Effect of a Deleterious Mutation in the ACT7 Gene on Hormone-Induced Callus Formation. Mutant samples are shown in the left panels and wild-type samples are shown in the right panels. (A) to (G) Young cotyledons of almost similar size before ([A] and [B]) and after 7 days ([C] and [D]) and 21 days ([E] to [G]) of hormone treatment. (F) and (G) show enlarged cotyledon-derived calli from (E). (H) and (I) Leaf explants after 21 days of incubation on CIM. Similar sized leaves were used for callus regeneration. (J) to (L) Root explants after 21 days of incubation on CIM. Bars in (A) to (D) = 1 mm; bars in (E) and (J) = 10 mm; bars in (F), (G), (H), (I), (K), and (L) = 5 mm.

Figure 7.

The ACT7 Protein Is Essential for Normal Callus Formation. Protein gel blot analysis of leaf callus extracts from the act7-1 mutant and wild-type (WT) plants with the general antibody MAbGPa and the subclass-specific antibodies MAb13a, MAb45a, and MAb2345a.

Figure 8.

Complementation of act7-1 Plants with the ACT7 Gene Sequence Reverses the Slow Regeneration of Callus to the Normal Wild-Type (WT) Level. Forty milligrams of young leaves was incubated on CIM, and after 21 days the fresh weight of the leaf explants was measured. Comp1 and Comp2 represent two independent complemented lines. Note that the callus regenerated from act7-1 mutant leaves had approximately 30% less weight than the wild-type leaf callus. Leaves from the complemented plants and act2-1 mutants produced callus almost equal to the wild-type leaves. The mean values of three different experiments with standard errors are shown.

Figure 9.

Callus Regeneration Is Severely Affected in the act2-1 act7-1 Double Mutant. (A) Ten-day-old act2-1 act7-1 double mutant seedling grown on Murashige and Skoog (1962) (MS) medium containing 3% Suc. (B) Ten-day-old wild-type seedling grown on similar medium. (C) and (D) The double mutant (C) and wild-type (D) cotyledons after 18 days of incubation on CIM. (E) and (F) Root explants of double mutant (E) and wild-type (F) plants after 25 days of incubation on CIM. Bars in (A) to (D) = 1 mm; bars in (E) and (F) = 5 mm.