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Figure 1.  Phenotypic characterizations of MIR319a over-expression transgenic polar lines. (a) Leaves from MIR319a over-expression transgenic lines and WT. The 2nd, 4th, 6th, 8th, and 10th represent the second, fourth, sixth, eighth and tenth leaves counted from the apical bud of MIR319a over-expression transgenic poplar plants, respectively. (b) The expression levels of miR319a in WT and 14 transgenic lines measured by qRT-PCR with three technical replicates.

Figure 2.  The vascular bundle and bundle sheath cells on microscopic leaf cross sections stained with phloroglucinol HCl in MIR319a over-expression transgenic lines and WT. The leaves were from the second, sixth and tenth stem nodes, respectively. The photos were taken at 10X, 40X and 100X. Bars in a−f: 200 μm, g−l: 50 μm, m−r: 20 μm. x, p and bs represent xylem, phloem and bundle sheath, respectively.

Figure 3.  Phylogenetic tree constructed with the protein sequences of 93 putative target genes of miR319a in Populus trichocarpa, 5 from Arabidopsis and 2 from rice. A neighbor-joining (NJ) method in MEGA7.0 was used to generate the tree. The tree consists of 3 distinct clades. The proteins whose genes were differentially expressed genes (DEGs) are highlighted in red.

Figure 4.  Expression levels of MEE35/TCP4, TCP2 and TCP2-1 determined by qRT-PCR in MIR319a over-expression transgenic leaves and WT.

Figure 5.  Three-layered hierarchical gene regulatory network under the control of miR319a in the leaves of P. alba × P. glandulosa. The green circle represents the leaf development gene, blue circles represent phloem or xylem histogenesis genes, the purple circle represents the phyllome development gene, gray circles represent xylem development genes, the red circle represents the lignin biosynthesis gene, and yellow circles represent photosynthesis genes. The gene IDs are provided in Supplemental Table 5.