Organ-specific phytohormone synthesis in two Geranium species with antithetical responses to far-red light enrichment

Abstract

Plants growing in high densities experience a reduced red (R) to far-red (FR) light ratio and shade-intolerant species respond with accelerated elongation growth to reach the top of the canopy: the shade avoidance syndrome (SAS). FR-enriched light inactivates phytochrome photoreceptors, which results in subsequent action of several plant hormones regulating growth. SAS is adaptive for shade-intolerant plants, but is suppressed in shade-tolerant plant species. Inspired by a previously published transcriptome analysis, we use two species of the genus Geranium here to study the involvement of auxin, brassinosteroids (BRs), and gibberellins (GAs) in supplemental FR-induced elongation growth. G. pyrenaicum, a shade-avoiding species, strongly induces auxin and gibberellin levels, but not BR, in elongating petioles. We show that, in this species, FR light perception, hormone synthesis, and growth are local and restricted to the petiole, and not the leaf lamina. Using chemical hormone inhibitors, we confirm the essential role of auxin and GAs in supplemental FR-induced elongation growth. Shade-tolerant G. robertianum does not display the change in hormone levels upon FR light enrichment, resulting in the lack of a shade avoidance response.

Keywords: Geranium; auxin; brassinosteroids; gibberellins; phytochrome signaling; shade avoidance.

Figure 1

Geranium pyrenaicum and G. robertianum transcriptionally induce phytohormone‐related processes in FR‐enriched light. Heat map presenting (over‐) representation of Gene Ontology terms associated with auxin‐, brassinosteroid‐, and gibberellin‐related processes among up‐ and downregulated OMCL groups after 2 hr or 11.5 hr of FR‐enriched (R:FR 0.2) light in G. pyrenaicum and G. robertianum. Terms are clustered according to the different hormones. Data from the RNA sequencing study published in Gommers et al., 2017. Colors represent ‐Log of the p‐value, and significantly overrepresented terms (‐Log(p) > 2) are marked with asterisks

Figure 2

Expression patterns of gibberellin‐, brassinosteroid‐, and auxin‐associated OMCL groups in Geranium pyrenaicum and G. robertianum after 2 or 11.5 hr of FR‐enriched light. Log2‐transformed fold changes (WL + FR/WL) of OMCL families subtracted from the GO categories presented in Figure 1. Data from the RNA sequencing study published in Gommers et al., 2017. Asterisks mark significant induction/suppression (p < 0.01)

Figure 3

FR light enrichment affects hormone synthesis‐ and signaling‐associated gene expression in Geranium pyrenaicum, but less so in G. robertianum. Expression of Geranium orthologues of TAA1, an AUX/IAA,SAUR50,BR6OX1,BZR1, and GA20OX2 in G. pyrenaicum (upper graphs) and G. robertianum (lower graphs) petioles upon 2 or 11.5 hr of FR light enrichment (WL + FR). Data are relative to the expression of a reference gene (orthologue of AT3G57890) and the WL control at 2 hr and represent means ± SE, n = 3 biological replicates. Asterisks indicate a significant difference between WL and WL + FR at the same time point, Student's t test, p < 0.05

Figure 4

FR‐enriched light affects hormone levels in the Geranium pyrenaicum petiole, but not in the lamina, nor G. robertianum. Free indole‐3‐acetic acid (IAA), brassinolide (BL), and gibberellin A₁ (GA ₁) levels (pg/mg fresh weight) in G. pyrenaicum and G. robertianum petioles (plain bars) and lamina (striped bars) exposed to 2 or 11.5 hr of control white light (WL; R:FR 1.8) or WL supplemented with far‐red light (WL + FR; R:FR 0.2). Data represent means ± SEM, n = 3 biological replicates. Asterisks represent significant differences in WL + FR compared to WL (Student's t test, p < 0.05). ND marks samples where no hormone was detected

Figure 5

FR light illumination of the Geranium petiole, and not the lamina, induces elongation. Petiole elongation (mm 24 hr ⁻¹) of G. pyrenaicum and G. robertianum plants grown in control white light (WL), WL supplemented with FR (WL + FR), or WL supplemented with a local FR treatment to only the petiole (WL + FR _petiole) or the lamina (WL + FR _lamina) for 24 hr. The treated leaf is measured. Data represent means ± SEM, n = 8 biological replicates. Different letters represent significant differences (1‐way ANOVA, p < 0.05)

Figure 6

Auxin, brassinosteroid, and gibberellin induce growth in Geranium pyrenaicum and G. robertianum petioles. G. pyrenaicum (a–c) and G. robertianum (d–f) petiole elongation (mm 24 hr ⁻¹) after treatment with different concentrations of 1‐naphthaleneacetic acid (1‐NAA; 0.1, 1, 10, 50, 100, 500 μM; a & d), 24‐epibrassinolide (epi BL; 10, 50, 100 μM; b,e), or gibberellic acid (GA ₃; 10, 50, 100 μM; c,f), which were sprayed on the petiole and lamina (total 250 μL/leaf). Data represent means ± SEM, n = 10 (1‐NAA) or n = 5 (epi BL and GA ₃) biological replicates. Asterisks represent significant differences with the mock control (0 μM) (Student's t test, p < 0.05)

Figure 7

Inhibition of auxin or gibberellins, but not brassinosteroids, represses supplemental FR light‐induced elongation in Geranium pyrenaicum. Cotyledon petiole length of G. pyrenaicum seedlings after five (a) or four (b–c) days in control white light (WL; R:FR 1.8) or WL supplemented with far‐red light (WL + FR; R:FR 0.2), combined with different concentrations of hormone inhibitors: (a) α‐(phenylethyl‐2‐one)‐IAA (PEO‐IAA; inhibitor of auxin perception), 1‐N‐naphthylphthalamic acid (NPA; inhibitor of auxin transport), (b) brassinazole (BRZ; inhibitor of brassinosteroid synthesis), or (c) paclobutrazol (PAC; inhibitor of gibberellin synthesis). Chemicals were added to a medium, to which plants were transferred 24 hr prior to the start of the light treatments. Data represent means ±SEM, n = 6–10. Asterisks mark significant differences compared to mock in the same light treatment (Student's t test, p < 0.05)

Figure 8

Graphic summary. When plants grow in dense communities, the R:FR light ratio decreases through R absorption and FR reflection, which is perceived in the petioles and causes a local increase in auxin and gibberellin (GA), but a reduction in brassinosteroid (BR) in shade‐avoiding plant species Geranium pyrenaicum (left). These changing hormone levels regulate the elongation of petioles, which facilitates shade escape. When shade‐tolerant species G. robertianum (right) is exposed to FR‐enriched light, this does not affect hormone synthesis or signaling, which results in no elongation response in its petioles