Transcription Profile of Auxin Related Genes during Positively Gravitropic Hypocotyl Curvature of Brassica rapa

Abstract

Unlike typical negative gravitropic curvature, young hypocotyls of Brassica rapa and other dicots exhibit positive gravitropism. This positive curvature occurs at the base of the hypocotyl and is followed by the typical negative gravity-induced curvature. We investigated the role of auxin in both positive and negative hypocotyl curvature by examining the transcription of PIN1, PIN3, IAA5 and ARG1 in curving tissue. We compared tissue extraction of the convex and concave flank with Solid Phase Gene Extraction (SPGE). Based on Ubiquitin1 (UBQ1) as a reference gene, the log (2) fold change of all examined genes was determined. Transcription of the examined genes varied during the graviresponse suggesting that these genes affect differential elongation. The transcription of all genes was upregulated in the lower flank and downregulated in the upper flank during the initial downward curving period. After 48 h, the transcription profile reversed, suggesting that the ensuing negative gravicurvature is controlled by the same genes as the positive gravicurvature. High-spatial resolution profiling using SPGE revealed that the transcription profile of the examined genes was spatially distinct within the curving tissue. The comparison of the hypocotyl transcription profile with the root tip indicated that the tip tissue is a suitable reference for curving hypocotyls and that root and hypocotyl curvature are controlled by the same physiological processes.

Keywords: Brassica rapa; Solid Phase Gene Extraction (SPGE); germination; gravitropism; hypocotyl; root.

Figure 1

(A) Extraction of mRNA using Solid Phase Gene Extraction: Oligo-dT15 functionalized stainless-steel needles were inserted into the upper and lower side of horizontally placed hypocotyls and the root tip of Brassica rapa seedlings. The hybridization between the dT and poly A tail of the mRNA was accomplished in 60 s. (B) Drawings that represent hypocotyl curvature during the experiments. (C) Sampling of the upper and lower flank of hypocotyl tissue.

Figure 2

Absolute Cq values for UBQ1 obtained by tissue extraction (solid lines) and SPGE (patterned lines). The top flank showed reduced (higher Cq values) transcription compared to the bottom flank. The numbers 1–6 correspond to locations in Figure 1. Mean ± SE, n = 9.

Figure 3

The ratio of Cq values of UBQ1 in bottom vs. top flank obtained from hypocotyl tissue (solid line) and SPGE (dashed line) after horizontal reorientation of B. rapa seedlings. Time = 0 refers to vertical position. Transcription of UBQ1 did not change significantly after reorientation (p = 0.75); mean ± SE, n = 9.

Figure 4

Absolute Cq values of the investigated genes PIN1, PIN3, IAA5 and ARG1 obtained after extracting tissue of the left and right flank of a vertically grown B. rapa seedlings. Mean ± SE, n = 9.

Figure 5

Log (2) fold change in transcription level of PIN1, PIN3, IAA5 and ARG1 in tissue samples of the upper (black) and lower (red) flank of hypocotyls of horizontally reoriented Brassica rapa seedlings four and 11 h after reorientation relative to the transcription of UBQ1. The transcription was upregulated in the bottom flank and downregulated in upper flank. The least significant difference (LSD) is based on the error of all measurements per panel. Mean ± SE, n = 9.

Figure 6

Log (2) fold change in transcription level of PIN1, PIN3, IAA5 and ARG1 after Solid Phase Gene Extraction (SPGE) of curving Brassica rapa hypocotyls relative to UBQ1 values. Probe 1 (apical end of curvature zone), 2 (center), and 3 (basal end, at root/shoot junction) sampled the upper flank (black lines) and probes 4, 5 and 6 sampled the corresponding positions along the bottom flank (red lines), see Figure 1. The transcription profiles are shown for 4, 11 and 48 h after horizontal reorientation. Positive and negative values indicate up- and downregulation, respectively. The least significant difference (LSD) is based on the error of all measurements per panel. Mean ± SE, n = 9.

Figure 7

Comparison of transcription profiles of PIN1, PIN3, IAA5 and ARG1 in B. rapa seedlings in tissue after four and 11 h of reorientation (upper panel) and SPGE extraction (lower panel) after four, 11 and 48 h reorientation. The data for SPGE top and bottom refers to the average values of the combined probes 1, 2, 3 and 4, 5, 6, respectively. All values are shown as fold change based on UBQ1. Mean ± SE, n = 9.

Figure 8

The transcription profile of PIN1, PIN3, IAA5 and ARG1 in the root tip after horizontal reorientation of B. rapa seedlings. Data were tissue (red) or SPGE (black) sampling of the 2 mm tip region. The transcripts were referenced to UBQ1. The least significant difference (LSD) was calculated separately for tissue and SPGE samples. Mean ± SE, n = 3.