Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis

Abstract

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.

Figure 1

Expression of PFN proteins and transcript in WT and transgenic seedlings. Proteins were separated by isoelectric focusing followed by SDS-PAGE. Affinity antibodies against PFN were used for the immunostaining. In A, B, and C, the white arrow indicates PFN-1 and the black arrow represents other members of the PFN family. RNA gel blots were hybridized to an in vitro transcribed antisense PFN-1 RNA. A, WT, 100 μg. B, PFN-O-3, 100 μg. C, PFN-U-6, 100 μg. D, PFN transcript levels in WT and transgenic plants. Each lane contained 30 μg RNA. Black arrow, Endogenous PFN mRNA; white arrow, transgenic PFN-1 mRNA. Lane 1, WT; lane 2, PFN-O-3; lane 3, PFN-O-6; lane 4, PFN-U-1; lane 5, PFN-U-3; lane 6, PFN-U-6. E, 18S rRNA levels in WT and transgenic plants. RNA gel blot from D was stripped and rehybridized with 18S rDNA lanes 1 through 6 same as D.

Figure 2

Phenotypes of PFN-O and PFN-U transgenic seedlings. A, WT, 10 d in white light at 22°C. B, PFN-O-3, 10 d in white light at 22°C. C, PFN-U-6, 10 d in white light at 22°C. D, WT, 10 d in dark at 22°C; average hypocotyl length = 1.36 ± 0.6 cm (n = 20). E, PFN-O-3, 10 d in dark at 22°C; average hypocotyl length = 1.40 ± 1.7 cm (n = 40). F, PFN-U-6, 10 d in dark at 22°C; average hypocotyl length = 0.54 ± 0.1 cm (n = 40). G, WT, 28 d in dark at 4°C. H, PFN-O-3, 28 d in dark at 4°C. I, PFN-U-6, 28 d in dark at 4°C. Bars in A, B, and C = 0.2 cm; bars in D, E, and F = 0.5 cm; bars in G, H, and I = 0.2 cm.

Figure 3

Hypocotyl phenotypes of transgenic seedlings. Seedlings were grown for 10 d in the dark and sections were made through the mid-portions of the hypocotyls. A, WT, cross section. B, PFN-O-3, cross section. C, PFN-U-6, cross section. D, WT, longitudinal section. Average length of cortical cells = 567 ± 131 μm; average width of cortical cells = 27.45 ± 7.23 μm; n = 80. E, PFN-O-3, longitudinal section. Average length of cortical cells = 569 ± 147 μm; average width of cortical cells = 32.75 ± 7.59 μm; n = 80. F, PFN-U-6, longitudinal section. Average length of cortical cells = 266 ± 71 μm; average width of cortical cells = 39.77 ± 10.27 μm; n = 80. G, WT, whole hypocotyl; width = 172.7 ± 12.0 μm (n = 5). H, PFN-O-3, whole hypocotyl; width = 205.3 ± 3.5 μm (n = 5). I, PFN-U-6, whole hypocotyl; width = 250.8 ± 27.0 μm (n = 5). Arrows in C point to the collapsed epidermal cells and arrowheads in C and F indicate some structures in the epidermal and cortex cells. Arrows in I show the swollen region of A, B, and C; same magnification. Bar in C = 0.5 mm; D, E, and F, same magnification; bar in F = 0.1 mm; G, H, and I, same magnification; bar in I = 0.1 mm.

Figure 4

SEM analysis of hypocotyl, petiole, and cotyledon surfaces of WT and transgenic seedlings. Hypocotyl and petiole specimens were prepared from the mid-portions of 10-d-old light- and dark-grown seedlings. A, Hypocotyl of light-grown WT. B, Hypocotyl of light-grown PFN-O-3. C, Hypocotyl of light-grown PFN-U-6. D, Cotyledon petiole of light-grown WT. E, Cotyledon petiole of light-grown PFN-O-3. F, Cotyledon petiole of light-grown PFN-U-6. G, Cotyledon surface of light-grown WT. H, Cotyledon surface of light-grown PFN-O-3. I, Cotyledon surface of light-grown PFN-U-6. J, Hypocotyl of etiolated WT. K, Hypocotyl of etiolated PFN-O-3. L, Hypocotyl of etiolated PFN-U-6. Bars = 100 μm.

Figure 5

Phenotypes of mature PFN-O and PFN-U plants grown in the light. Seedlings were grown for 20 d in Petri plates and then transferred into soil. A, 15 d after transfer. B, 25 d after transfer. C, 35 d after transfer. D, 45 d after transfer. For A through D, left, WT; middle, PFN-O-3; right, PFN-U-6. Bars = 4 cm.

Figure 6

GUS staining pattern of PFN-1-GUS transgenic seedlings and root phenotypes of PFN-O seedlings. A, GUS expression in etiolated seedlings. B, GUS expression pattern in 20-d-old light-grown seedlings. C, GUS activity in the root and root hairs of 20-d-old light-grown seedlings. D and E, GUS staining of the root elongation zone of 20-d-old light-grown seedlings. F, Roots of 5-d-old light-grown WT seedlings. G, Roots of 5-d-old light-grown PFN-O-3 seedlings. H, Root hairs of 5-d-old light-grown WT seedlings. I, Root hairs of 5-d-old light-grown PFN-O-3 seedlings. Bar in A = 0.2 mm; B = 0.2 mm; C = 0.05 mm; D = 0.05 mm; E = 0.01 mm; F and G = 0.2 mm; H and I = 0.05 mm.