A Role for Auxin in Triggering Lamina Outgrowth of Unifacial Leaves

Abstract

A common morphological feature of typical angiosperms is the patterning of lateral organs along primary axes of asymmetry-a proximodistal, a mediolateral, and an adaxial-abaxial axis. Angiosperm leaves usually have distinct adaxial-abaxial identity, which is required for the development of a flat shape. By contrast, many unifacial leaves, consisting of only the abaxial side, show a flattened morphology. This implicates a unique mechanism that allows leaf flattening independent of adaxial-abaxial identity. In this study, we report a role for auxin in outgrowth of unifacial leaves. In two closely related unifacial-leaved species of Juncaceae, Juncus prismatocarpus with flattened leaves, and Juncus wallichianus with transversally radialized leaves, the auxin-responsive gene GLYCOSIDE HYDROLASE3 displayed spatially different expression patterns within leaf primordia. Treatment of J. prismatocarpus seedlings with exogenous auxin or auxin transport inhibitors, which disturb endogenous auxin distribution, eliminated leaf flatness, resulting in a transversally radialized morphology. These treatments did not affect the radialized morphology of leaves of J. wallichianus. Moreover, elimination of leaf flatness by these treatments accompanied dysregulated expression of genetic factors needed to specify the leaf central-marginal polarity in J. prismatocarpus. The findings imply that lamina outgrowth of unifacial leaves relies on proper placement of auxin, which might induce initial leaf flattening and subsequently act to specify leaf polarity, promoting further flattening growth of leaves.

Figure 1

Auxin-inducible GH3c as a useful marker of auxin physiology. (A–H) In situ localization of GH3c transcript (A–C, E–G) and immunolocalization of IAA (auxin) (D and H) in transverse sections of J. prismatocarpus (A–D) and J. wallichianus (E–H) shoot apices through P2 leaf blades or sheaths without treatment (A, D, E, and H), with endogenous auxin depletion (B and F), or IAA administration after auxin depletion (C and G). Black arrowheads indicate localization, and white arrowheads indicate lack of expression in domains corresponding to the GH3c-positive domains in untreated samples. cv, central large vascular bundle. Scale bars are 200 µm in all panels. More than five independent shoot apices with the indicated treatments were analyzed for GH3c localization in each species, and all gave similar results. Four independent shoot apices were subjected to IAA immunolocalization, and all gave similar results. (I) RT-qPCR results showing the relative quantity of GH3c mRNA normalized to that of TUBULIN mRNA in auxin-depleted J. prismatocarpus shoot apices treated with IAA or 2,4-D and without (con.). Shown are the means ± SD of three independent experiments. ^*P < 0.05; ^**P < 0.005; ^***P < 0.001. One-way ANOVA was carried out to calculate P-values. (J) Putative promoter of the GH3c gene in J. prismatocarpus (Jp) and J. wallichianus (Jw), showing two auxin-response elements within. Numbers in parentheses are the genomic positions of the indicated regions relative to the transcription initiation site (+1).

Figure 2

Distinct GH3c localization in leaf primordia in J. prismatocarpus and J. wallichianus. (A–E) In situ localization of GH3c transcript in J. prismatocarpus shoot apices. (A–D) Transverse sections of shoot apices through a P1 leaf sheath (A), a P2 leaf sheath (B), a P2 leaf blade (C), and a P3 leaf blade (D), showing GH3c expression (arrowheads). (E) Median longitudinal section through the SAM and a P2 leaf primordium, showing GH3c expression both at a side adjacent to the SAM (arrow) and a side furthest from the SAM (arrowhead). (F–J) In situ localization of GH3c transcript in J. wallichianus shoot apices. (F–I) Transverse sections of shoot apices through a P1 leaf blade (F), a P2 leaf sheath (G), a P2 leaf blade (H), and a P3 leaf blade (I), showing GH3c expression (arrowheads). Note the distinct GH3c distribution between J. prismatocarpus and J. wallichianus. (J) A median longitudinal section through the SAM and a P2 leaf primordium, showing GH3c expression only at a central vascular bundle (arrowheads). White arrow indicates loss of GH3c expression at a side adjacent to the SAM. bl, leaf blade; sh, leaf sheath; cv, central large vascular bundle. Plastochron numbers of leaf blades or sheaths are indicated in parentheses. Scale bars are 200 µm in all panels. More than five independent shoot apices at each stage were analyzed for GH3c localization in each species, and all gave similar results.

Figure 3

Disturbance of endogenous auxin distribution eliminates leaf flatness in J. prismatocarpus. (A–I) Transverse sections of J. prismatocarpus mature leaf blades at 28 d after sowing (A, D, and G), shoot apices through P2 leaf blade primordia (B, E, and H), or through P3 leaf blade primordia (C, F, and I). Samples had been treated with mock (A–C), 2,4-D (D–F), or NPA (G–I). (J) Time-course changes in flatness of J. prismatocarpus mature leaf blades during cultivation of seedlings with the indicated treatments. Shown are the means ± SD from five independent seedlings. (K–S) Transverse sections of J. wallichianus mature leaf blades at 28 d after sowing (K, N, and Q), shoot apices through P2 leaf blade primordia (L, O, and R), or through P3 leaf blade primordia (M, P, and S). Samples had been treated with mock (K–M), 2,4-D (N–P), or NPA (Q–S). (J) Time-course changes in flatness of J. wallichianus mature leaf blades during cultivation of seedlings with the indicated treatments. Shown are the means ± SD from five independent seedlings. bl, leaf blade. Plastochron numbers of leaf blades are indicated in parentheses. Scale bars are 200 µm. Transverse sections of ˃4 independent shoot apices with the indicated treatments were analyzed for each species, and all gave similar results.

Figure 4

Disturbed GH3c distribution caused by 2,4-D- or NPA is associated with eliminated leaf flatness in J. prismatocarpus. Transverse sections of shoot apices of J. prismatocarpus (A–F) and J. wallichianus (G–L), showing in situ localization of GH3c in P2 leaf blade or sheath primordia. Samples had been treated with mock (A, B, G, and H), 2,4-D (C, D, I, and J), or NPA (E, F, and K, and L). bl, leaf blade; sh, leaf sheath. Plastochron numbers of leaf blades or sheaths are indicated in parentheses. Scale bars are 200 µm in all panels. Six independent J. prismatocarpus shoot apices and three J. wallichianus shoot apices with the indicated treatments were analyzed for GH3c localization, and all gave similar results.

Figure 5

Disturbance of endogenous auxin distribution accompanies dysregulated expression of DL and PRSb in leaf primordia of J. prismatocarpus. Transverse sections of shoot apices in J. prismatocarpus, showing in situ localization of DL in the central domains of P2 leaf blade primordia (A, D, and G), DL in the secondary central domains of P3 leaf blade primordia (B, E, and H), and PRSb in margin-like domains of P3 leaf blade primordia (C, F, and I). Samples had been treated with mock (A–C), 2,4-D (D–F), or NPA (G–I). Black arrows and arrowheads indicate expression, and white arrows and arrowheads indicate loss of DL or PRSb expression in domains corresponding to the DL- or PRSb-positive domains in mock-treated J. prismatocarpus samples. bl, leaf blade; cv, central large vascular bundle. Plastochron numbers of leaf blades are indicated in parentheses. Scale bars are 200 µm in all panels. More than four independent shoot apices with the indicated treatments were analyzed for localization of DL and PRSb transcripts, and all gave similar results.

Figure 6

Proposed roles for auxin in guiding lamina outgrowth of flattened unifacial leaves. (A) An auxin maximum (red circle) first appears in a P1 leaf primordium at a side far from the SAM. (B) An auxin maximum at the subsequent P2 stage (red circle) is needed to activate leaf lamina outgrowth in concert with DL (black arrow). (C) The pairwise central auxin maxima (blue circles) define the sites to be specified into the presumptive central domains. In addition, auxin maxima detected at the tips of a flattened leaf primordium (red circles) serve to specify the presumptive marginal domains. (D) Auxin maxima correspond to the DL (black arrowheads) and the PRSb (green circles) expression sites, which further establish the central-marginal polarity of a leaf.