The HD-ZIP II Transcription Factors Regulate Plant Architecture through the Auxin Pathway

Abstract

The homeodomain-leucine zipper (HD-ZIP) family transcription factors play important roles in plant growth and development. However, the underlying mechanisms remain largely unclear. Here we found that ATHB2, encoding a HD-ZIP transcription factor, is an early auxin responsive gene. Phenotypic analyses show that overexpression of ATHB2 impairs plant architecture, including reduced plant height and small leaves, and also reduces auxin response in leaves when grown in soil. Simultaneously, the seedlings with chemical induction of ATHB2 exhibit abnormal root gravitropism, a typical auxin-related phenotype. We further show that the auxin response pattern is altered in roots of the inducible ATHB2 seedlings. Consistently, the transcript levels of some auxin biosynthetic and transport genes are significantly decreased in these transgenic seedlings. Further, protein and promoter sequence analyses in common wheat showed that the HD-ZIP II subfamily transcription factors have highly conserved motifs and most of these encoding gene promoters contain the canonical auxin-responsive elements. Expression analyses confirm that some of these HD-ZIP II genes are indeed regulated by auxin in wheat. Together, our results suggest that the HD-ZIP II subfamily transcription factors regulate plant development possibly through the auxin pathway in plants.

Keywords: ATHB2; Arabidopsis; HD-ZIP; auxin; wheat.

Figure 1

Expression patterns of the ATHB2 subset genes. (A–F) Expression analyses of ATHB2, HAT1, HAT2, HAT3, ATHB4 and 1AA19 in response to auxin treatment. The 6 day old wild-type (Col-0) seedlings were treated with 10 μM IAA for different time points. The ACTIN7 gene was used as an internal reference. The qRT-PCR results were performed for three biological replications and similar results were observed. Representative qRT-PCR results with three technical replicates were shown. Error bars denote ± SD.

Figure 2

Morphological phenotype of 35S:ATHB2-FLAG transgenic lines. (A) Immunoblotting analysis showing the ATHB2-FLAG protein levels in the 35S:ATHB2-FLAG transgenic plants. The 6 day old seedlings of Col-0 and 35S:ATHB2-FLAG transgenic plants were harvested for immunoblotting analysis. ACTIN was used as a loading control. 1# and 2# represent two independent transgenic lines of 35S:ATHB2-FLAG. The data are representative of three independent experiments. (B and C) Overview of the Col-0 and 35S:ATHB2-FLAG transgenic lines at the adult stage. (B) Scale bars, 4cm. Leaf morphology of the Col-0 and 35S:ATHB2-FLAG transgenic lines at the adult stage. (C) Scale bars, 2 mm. The 4-week-old 35S:ATHB2-FLAG transgenic plants grown under normal growth conditions were used for phenotypic analyses. (B and C) Two independent transgenic lines were used for phenotype observation. The images are representative of three independent experiments. (D) Expression patterns of DR5:GUS in the leaves of DR5:GUS and DR5:GUS/35S:ATHB2-FLAG plants. The leaves of 3-week-old DR5:GUS and DR5:GUS/35S:ATHB2-FLAG plants were used for GUS activity analyses. The images are representative of three independent experiments.

Figure 3

Root gravitropic phenotype of β-estradiol-inducible ATHB2-overexpression transgenic lines. (A) qRT-PCR analysis showing the β-estradiol-induced expression pattern of ATHB2 in the iATHB2 lines. The ACTIN gene was used as an internal reference. 1# and 2# represent two independent transgenic lines of iATHB2. The qRT-PCR results were performed for three biological replications and similar results were observed. Representative qRT-PCR results with three technical replicates were shown. Error bars denote ± SD. (B) Root gravitropic phenotypes of the iATHB2 transgenic lines. Seedlings of the Col-0 and inducible ATHB2 overexpression plants (iATHB2) were grown on 1/2 MS medium with or without 10 μM β-estradiol for six days. Two independent transgenic lines were used for phenotypic observation. The images are representative of three independent experiments.

Figure 4

Asymmetric auxin response was observed in the root tips of iATHB2 plants. The spatial expression pattern of the DR5rev:GFP auxin responsive reporter in the root tips of 6-day-old DR5rev:GFP/iATHB2 plants grown on the medium with or without 10 μM β-estradiol. The images are representative of three independent experiments. Roots were stained with propidium iodide (red). Arrowheads indicate the asymmetric auxin response in the root tips. Scale bars = 50 μm. Arrowheads indicate the asymmetric auxin response of DR5rev:GFP/iATHB2 with 10 μM β-estradiol treatment.

Figure 5

Overexpression of ATHB2 reduced the expression levels of some auxin biosynthetic and transport genes. (A–F) Expression patterns of YUC2, YUC8 and several PINs genes in the iATHB2 transgenic lines. The different 6-day-old iATHB2 transgenic lines were treated with or without 10 μM β-estradiol for 2 h. The ACTIN7 gene was used as an internal reference. The shown data are results of the representative iATHB2 2# transgenic line. The qRT-PCR results were performed for three biological replications and similar results were observed. Representative qRT-PCR results with three technical replicates were shown. Error bars denote ± SD. * p < 0.05, ** p < 0.01, Student’s t test. No significant difference is shown by n.s.

Figure 6

Schematic diagrams of the conserved domain structure of wheat HD-ZIP II subfamily proteins. Red box indicates the homeobox domain (HD) and blue box indicates the adjacent leucine zipper (LZ) motif, respectively.

Figure 7

Putative motifs of the wheat HD-ZIP II subfamily proteins using the MEME program. The different conserved motifs are marked by different colors.

Figure 8

The “TGTCTC” elements in the 3 Kb genomic regions upstream of coding regions are indicated by blue box.

Figure 9

Several wheat HD-ZIP II subfamily genes were regulated by auxin. (A–D) Auxin-induced expression pattern of TaHDZ19-3A/3B/3D, TaHDZ20-1A/1B/1D, TaHDZ21-2A/2B/2D and TaHDZ23-7A/7D by qRT-PCR. The 4-day-old wheat seedlings were treated with 10 μM IAA for 4 h. The TaGAPDH gene was used as an internal reference. The qRT-PCR results were performed for three biological replications and similar results were observed. Representative qRT-PCR results with three technical replicates are shown. Error bars denote ± SD. ** p < 0.01, Student’s t test. No significant difference is shown by n.s.