Key auxin response factor (ARF) genes constraining wheat tillering of mutant dmc

Abstract

Tillering ability is a key agronomy trait for wheat (Triticum aestivum L.) production. Studies on a dwarf monoculm wheat mutant (dmc) showed that ARF11 played an important role in tillering of wheat. In this study, a total of 67 ARF family members were identified and clustered to two main classes with four subgroups based on their protein structures. The promoter regions of T. aestivum ARF (TaARF) genes contain a large number of cis-acting elements closely related to plant growth and development, and hormone response. The segmental duplication events occurred commonly and played a major role in the expansion of TaARFs. The gene collinearity degrees of the ARFs between wheat and other grasses, rice and maize, were significantly high. The evolution distances among TaARFs determine their expression profiles, such as homoeologous genes have similar expression profiles, like TaARF4-3A-1, TaARF4-3A-2 and their homoeologous genes. The expression profiles of TaARFs in various tissues or organs indicated TaARF3, TaARF4, TaARF9 and TaARF22 and their homoeologous genes played basic roles during wheat development. TaARF4, TaARF9, TaARF12, TaARF15, TaARF17, TaARF21, TaARF25 and their homoeologous genes probably played basic roles in tiller development. qRT-PCR analyses of 20 representative TaARF genes revealed that the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc. Indole-3-acetic acid (IAA) contents in dmc were significantly less than that in Guomai 301 at key tillering stages. Exogenous IAA application significantly promoted wheat tillering, and affected the transcriptions of TaARFs. These data suggested that TaARFs as well as IAA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat.

Keywords: Auxin response factor; Expression profiles; IAA; Tillering; Wheat (Triticum aestivum L.).

Figure 1. The tiller micromorphology of Guomai 301 (left) and mutant dmc (right).

(A) The individual plants of Guomai 301 and mutant dmc in the field condition. (B) The seedlings of Guomai 301 and dmc at the three-leaf stage. (C) The seedlings of Guomai 301 and dmc at the over-winter stage; (D) The seedlings of Guomai 301 and dmc at the rising to jointing stage. (E) Tiller primordia of Guomai 301 and dmc at the three-leaf stage. (F) Tiller primordia of Guomai 301 and dmc at the over-winter stage. (G) Tiller primordia of Guomai 301 and dmc at the rising to jointing stage. MC: main culm; TP: tiller primordium; Scale bar: 10 cm (A); 2 cm (B–D); 1 cm (E–G).

Figure 2. The endogenous IAA contents in tiller primordia of Guomai301 and dmc.

S1: the three-leaf stage; S2: the five-leaf stage; S3: the over-winter stage. Asterisks indicate significant difference or highly significant difference between Guomai 301 and dmc in different stages.

Figure 3. The tiller number changes of Guomai 301 and dmc in response to IAA treatments.

T1-T6 of the x-axis indicated the sampling dates, and the tiller numbers were recorded every 7 days. T1 is the first sampling date which was the 18th day after IAA treatment. Asterisks indicate significant difference or highly significant difference between treated groups and control groups in different sampling dates, respectively.

Figure 4. Phylogenetic tree of ARF proteins from Arabidopsis, maize, rice and wheat.

The purple solid diamonds represent ARF proteins in Arabidopsis (AtARF); The green squares represent ARF proteins in maize (ZmARF); The blue deltas represent ARF proteins in rice (OsARF); The red solid circles represent ARF proteins in wheat (TaARF); The different colored sectors indicate different groups (or subgroups) of ARF proteins. The different colored arcs indicate different classes of ARF proteins.

Figure 5. Phylogenetic relationships, conserved protein motif patterns, domain patterns and gene structures of TaARFs.

(A) The phylogenetic tree of TaARF proteins. Clusters are indicated with different colors. (B) The motif compositions of TaARFs. The 1–10 motifs are displayed in different colored boxes, the scale at the bottom indicates the length of proteins. (C) The domain patterns of TaARFs, the B3 domains are highlighted in yellow, the auxin response domains are highlighted in green, and the AUX_IAA domain are highlighted in lilac. (D) Exon-intron structures of TaARFs, yellow boxes indicate 5′- and 3′- untranslated regions; green boxes indicate exons; black lines indicate introns.

Figure 6. The cis-acting elements in the promoters of TaARFs.

Growth-related cis-element: meristem expression regulation (CAT-box and CCGTCC motifs); hormone response-related cis-elements: abscisic acid response (ABRE), methyl jasmonate response (CGTCA-motif), salicylic acid response (TCA-element), gibberellic response (P-box), auxin response (TGA-element and AuxRR-core) and ethylene response (ERE).

Figure 7. Schematic diagram of the chromosome distribution and interchromosome relationships of TaARFs.

The grey lines indicate all duplicated gene pairs in wheat, the highlighted red lines indicate probably duplicated TaARF gene pairs.

Figure 8. Syntenic relationships of ARF genes between wheat and three representative species.

Gray lines in the background indicate the collinear blocks within wheat and other plant genomes, while the blue lines highlight the syntenic ARF gene pairs.

Figure 9. Expression profiles of TaARFs in various organs or tissues.

(A) Heatmap of expression profiles of TaARFs in various organs or tissues of Chinese Spring from the Wheat Expression Browser (http://www.wheat-expression.com/). (B) The heat map of expression profiles of TaARFs in tiller primordia of WT and dmc based on transcriptome data. Three biological replicates were set up in the mutant dmc (T01, T02 and T03) and WT (T04, T05 and T06), and each sample bulk of tiller primordia included more than 10 independent individuals.

Figure 10. QRT-PCR results of 20 TaARFs in the tiller primordia of WT and dmc at three tillering stages.

WT1, dmc 1: the three-leaf stage; WT2, dmc 2: the over-winter stage; WT3, dmc 3: the rising to jointing stage. Data were normalized to β-actin gene and vertical bars indicated standard deviation. Asterisks indicate significant difference or highly significant difference between Guomai 301 and dmc.

Figure 11. Expression profiles of 20 TaARFs in response to IAA treatment.

Data were normalized to β-actin gene and vertical bars indicated standard deviation. Asterisks indicate significant difference or highly significant difference between Guomai 301 and dmc.