Possible involvement of CS-ACS1 and ethylene in auxin-induced peg formation of cucumber seedlings

Abstract

Background and aims: Cucumber (Cucumis sativus) seedlings develop a peg on the concave side of the gravitropically bending transition zone between the hypocotyl and the root after seed germination. Peg initiation occurs in response to auxin when its levels in the concave side of the transition zone exceed a particular threshold through the graviresponse. Ethylene also plays an important role in peg formation, but its relationship to auxin in this event is not understood. Here, the role ethylene plays in auxin-induced peg formation is studied.

Methods: Peg formation of cucumber seedlings exposed to ethylene at different stages of growth or during exogenous auxin treatment was observed. In addition, ethylene evolution from the concave and convex sides of the transition zone was compared and their transcription of CS-ACS (1-aminocyclopropane-1-carboxylic acid synthase) genes was analysed by RT-PCR and in situ hybridization.

Key results: Seedlings treated with ethylene after peg initiation produced an enlarged peg, whereas ethylene treatment before peg initiation inhibited peg formation. Ethylene also promoted the development of the peg in the auxin-treated seedlings. Furthermore, the concave side of the transition zone at peg initiation produced more ethylene and CS-ACS1 mRNA than the convex side.

Conclusions: Since CS-ACS1 is an auxin-inducible gene, the greater abundance of auxin in the concave side of the transition zone causes peg initiation and increases CS-ACS1-mediated ethylene biosynthesis, which then facilitates peg development.

Fig. 1.

Seedling growth and peg formation as affected by exogenous ethylene in cucumber. (A–C) The 72-h-old cucumber seedlings grown in a horizontal position. (D–F) The longitudinal section of the transition zone of 72-h-old cucumber seedlings. The seedlings were grown for 72 h in air (A and D) or in the presence of 20 μL L⁻¹ ethylene (B and E) or the seedlings were treated with 20 μL L⁻¹ ethylene only from 24 h to 72 h (C and F) after imbibition. c, Cotyledons; h, hypocotyl; pr, primary root; lr, lateral root. The arrowhead indicates the peg, and the arrow labelled g indicates the direction of gravity. Scale bars: A–C = 5 mm; D–F = 500 μm.

Fig. 2.

Effect of ethylene on the peg formation induced by local IAA application on the transition zone of cucumber seedlings. The seedlings were grown in ambient air (A, C and E) or in the presence of 20 μL L⁻¹ ethylene (B, D and F) until 24 h after imbibition, after which lanolin paste containing 0·6 mm IAA was applied to the convex side of the transition zone of seedlings grown in a horizontal position (A, B, E and F) or to one side of the transition zone of seedlings grown in a vertical position (C and D). The seedlings then continued to grow under the same air/ethylene conditions of the previous 24 h until 72 h after imbibition. Longitudinal sections were prepared from the transition zone of 72-h-old cucumber seedlings grown in a horizontal position (E and F). Black and white arrowheads indicate the peg and the site where IAA was applied, respectively. The arrow labelled g indicates the direction of gravity. Scale bars: A–D = 5 mm; E–F = 500 μm.

Fig. 3.

Ethylene evolution from cucumber seedlings grown in a horizontal position. (A) Analysis of the ethylene evolution over time from whole cucumber seedlings after imbibition. (B) Analysis of the ethylene evolution from the concave and convex sides of the transition zone of 24-h-old cucumber seedlings. The vertical bars indicate the standard deviations. The results are significantly different at P < 0·05 (Student's t-test).

Fig. 4.

Comparison of the mRNA accumulation of the CS-ACS genes in the concave and convex sides of the transition zone of 24-h-old cucumber seedlings grown in a horizontal position. (A) Schematic drawing of a 24-h-old seedling grown in a horizontal position showing the samples that were excised for quantitative RT-PCR. (B) Quantitative RT-PCR Southern blot analysis of CS-ACS expression. The RT-PCR products were amplified with the indicated cycle numbers.

Fig. 5.

Localization of CS-ACS1 mRNA in 24-h-old cucumber seedlings grown in a horizontal position. In situ hybridization with the antisense probe (A and B) and the sense probe (C). The arrowhead indicates the signals in the transition zone. (B) A higher magnification of the transition zone in (A). The arrow labelled g indicates the direction of gravity. Scale bars = 500 μm.

Fig. 6.

Northern blot analysis of the auxin-inducibility of CS-ACS1 and CsIAA1 in the transition zone. The transition zone was excised from 24-h-old seedlings that had not been treated (Non-treatment), had undergone auxin starvation for 1·5 h (Auxin starvation), had been treated with exogenous 10⁻⁴ m IAA for the indicated time after 2-h-auxin starvation, or had been incubated without IAA for 2 h after 2-h-auxin starvation (Incubation without IAA). The total RNAs were isolated from the transition zones, and each lane was loaded with 5 μg of total RNA, followed by hybridization with the CS-ACS1 and CsIAA1 RNA probes. EtBr indicates the ethidium bromide staining of ribosomal RNAs.