Figure 1.
The expression of miR156 and its targets is correlated with vegetative phase change in woody plants.
(A) Morphology of first two leaves of A. confusa. (B) Morphology of the first 8 leaves of A. colei. J = juvenile, T = transition, A = adult. (C) Juvenile and adult leaves from a single tree of E. globulus. (D) Juvenile and adult clones of H. helix (English ivy). (E) Juvenile and adult leaves of Q. acutissima. Scale bars indicate 2 cm. (F) One of the E. globulus trees from which the leaves used for expression analysis were harvested. (G) Q. acutissima tree from which the leaves used for expression analysis were harvested. (H) Blots of small RNA isolated from the leaves shown in Figure 1A–1E, hybridized with probes for miR156, miR157 and miR172; tRNAmet was used as a loading control. The H. helix blot represents RNA from shoot apices with leaves 1 cm or less in size. (I) qRT-PCR analyses of the expression of EglSPL3 and EglSPL9 in fully expanded juvenile and adult leaves of E. globulus. Expression was normalized to EglElF4, and then to the average expression in juvenile leaves. Shown are the averages of three technical replicates for samples from three trees (3 technical replicates×3 trees = 9 replicates per sample), ± s. e. m. Asterix = significantly different from juvenile, p<0.01, Student's t test.
Figure 2.
Vegetative phase change and miRNA expression in P. x canadensis.
(A) Leaf morphology. Scale bars indicate 2 cm for 1-month-old trees and 4 cm for the rest. (B) Transverse sections of petioles of 1-month- (left) and 1-year-old (right) trees. ab, abaxial. ad, adaxial. vb = vascular bundles. Scale bar indicates 200 µm. (C) Expression of miR156 and miR172, with U6 as loading control. (D) Expression of PcSPL3 and PcSPL9, measured by real-time RT-PCR, and normalized to PcACT. Error bars indicate standard deviation (s.d.). Asterix = significantly different from 1-month-old saplings, p<0.01, Student's t test.
Table 1.
Growth characteristics of 35S::MIR156 P. x canadensis plants.
Figure 3.
Spatial expression pattern of miR156 in P. x canadensis.
(A) Diagram illustrating the source of the leaf samples analyzed in 3B–3E. 2 cm leaf primordia were harvested from the shoot apex of 0.5 m, 2 m and 4 m tall shoots; fully expanded leaves were harvested from a branch (branch 1) of a 6 month-old tree, and 2 cm leaves or leaf primordia were harvested from branches located 2.5 m (branch 2) and 4 m (branch 3) from the base of a 1-year-old tree. Drawing is not to scale. (B) miRNA expression in 2 cm long leaf primordia from the shoot apices of trees of different heights. (C) Fully-expanded leaves from a single branch of a 6-month-old tree, numbered from the closest position to the trunk. (D) miRNA expression in the leaves illustrated in (C). (E) miR156 expression in 2 cm leaves or leaf primordia on branches located 2.5 m (branch 2), and 4 m (branch 3) from the base of the shoot.
Figure 4.
The phenotype of 35S::MIR156 P. x canadensis plants.
(A) qRT-PCR analysis of the expression of PcSPL3, PcSPL9, and PcFUL in 6-month-old plants, normalized to PcACT. Wild-type expression levels were normalized to 1. Asterix = significantly different from wild type, p<0.01, Student's t test. (B) Single branch from 6-month-old wild-type (WT) and 35S::MIR156 (#1) plants. Scale bar indicates 10 cm. (C) Leaves of 6-month-old wild-type and three independent 35S::MIR156 transgenic lines (#1 to #3). Scale bar indicates 5 cm. (D) Expression of miR156 and miR172. (E) Petiole sections of wild-type (left) and 35S::MIR156 (#1) (right). Scale bar indicates 200 µm. (F) Transverse sections of a major vein and the lamina of 6-month-old fully expanded leaves from wild-type (left) and 35S::MIR156 (#1) (right) plants. pp = palisade parenchyma. Scale bars indicate 50 µm. (G) Primary shoots of 2-month-old wild-type (left) and 35S::MIR156 (#1) (right) plants. Arrows indicate lateral shoots. Scale bars indicate 5 cm.