Genome-Wide Identification and Characterization of wALOG Family Genes Involved in Branch Meristem Development of Branching Head Wheat

Abstract

The branched spike phenotype is an important supernumerary spikelet trait of Triticum turgidum L. associated with the production of significantly more grains per spike, thereby offering a higher potential yield. However, the genetic basis of branch meristem (BM) development remains to be fully elucidated in wheat. TAW1, an ALOG (Arabidopsis LSH1 and Oryza G1) family gene, has been shown to function as a unique regulator in promoting BM development in rice. In this study, we found that the development pattern of the BMs of the branched spike in wheat was similar to the indeterminate BMs of rice. Moreover, phylogenetic analysis classified the ALOG genes into 12 groups. This family of genes was found to have evolved independently in eudicots and monocots and was evolutionarily conserved between wheat and rice as well as during wheat polyploidization. Furthermore, experiments revealed that TtALOG2-1A, a TAW1-homologous gene, plays a significant role in regulating the transition of indeterminate BM fate. Finally, large-scale RNA-sequencing studies and quantitative real-time polymerase chain reaction (qRT-PCR) experiments revealed that members of the TtALOGs may act upstream of the TtMADS22, TtMADS47, and TtMADS55 genes to promote indeterminate BM activities. Our findings further knowledge on BM development in wheat.

Keywords: branching head wheat; cis-acting regulatory elements; gene expression; phylogenetics; sequence diversification; wheat ALOG.

Figure 1

Morphology of the spikes of a normal head (nh) and the spikes of a branching head (bh). (A) Spike morphology of GAN-A631–left side (bh) and GAN-A1582–right side (nh). (B) The early double ridge (EDR) stage of bh. (C) The late double ridge (LDR) stage of bh. (D) The branch meristem (BM) development stage of bh and the red asterisk indicates the region of secondary spikelet meristem (SM) emergence. Compared with the glume primordia (GP) stage of nh, slight bulging can be observed at the location of the red asterisk in bh. (E) The SM initiation (SMI) stage of bh. (F) The EDR stage of nh. (G) The LDR stage of nh. (H) The GP stage of nh. (I) The lemma primordia (LP) stage of nh. Abbreviations: bm-l, branched-like meristem; b, bract; b-l, bract-like meristem; sm, spikelet meristem; gl, glume; l, lemma. Scale bars: (B–G), 100 µm; (H,I), 200 µm.

Figure 2

Phylogenetic relationship and conserved motifs of the selected ALOGs. A total of 86 ALOG proteins from Arabidopsis thaliana, Oryza sativa subsp. Japonica, Triticum urartu (AA), Aegilops tauschii (DD), Triticum turgidum ssp. Dicoccoides (AABB), and Triticum aestivum (AABBDD) were selected to construct the phylogenetic tree and identify the conserved motifs. The blue dots filled with red in the clades and the letters (a–l) show the 12 main groups. The genes written in bold font have known functional ALOG genes in Arabidopsis and rice. The names of species are abbreviated to two or three letters and detailed information is provided in Table 2. Each conserved motif is illustrated with a specific color and shape, and the distribution of the motifs corresponds to their positions. The first line labels the 143rd amino acid and the second line labels the 238th amino acid position in all sequences.

Figure 3

Heat map showing the expression profiling of the TtALOG, TtMADS22, TtMADS47, and TtMADS55 genes at the early stages of inflorescence development in GAN-A631 (bh). The relative expression levels are shown in the heat map by a gradient of color: blue/white/red (low to high). Abbreviations: EDR, the early double ridge stage; LDR, the late double ridge stage; BMD, the branch meristem development stage; SMI, the spikelet meristem-initiated stage. The heat map color key gradient ranging from global minimum (blue) −1 to global maximum (red) +1 is shown on the right side. Asterisks indicate the TtMADS22, TtMADS47, and TtMADS55 of GAN-A631.

Figure 4

Relative abundance of some ALOG homolog mRNAs as determined by quantitative real-time polymerase chain reaction (qRT-PCR) with samples from the spikes of GAN-A631 (bh) and GAN-A1582 (nh) at the early developmental stages of the spike. The values shown correspond to the mean values for three biological and three technical replicates. Spike developmental stages were assigned as follows, EDR, early double ridge stage; LDR, late double ridge stage; BMD, branch meristem development stage; SMI, spikelet meristem-initiated stage; GP, glume primordia stage; and LP, lemma primordia stage. The use of the bh and nh prefixes means that the samples came from GAN-A631 (bh) and GAN-A1582 (nh), respectively.

Figure 5

Analysis of the mutant ALOG proteins among wheat. The ALOG proteins were cloned from GAN-A631 (bh) and GAN-A1582 (nh) and their amino acid sequences were compared with those from the T. aestivum and T. turgidum ssp. Dicoccoides genomes. A conserved region and a nuclear localization signal are indicated with orange and green lines, respectively. GAN-A631 sequences: TtTAW1-1A and TtTAW1-1B. GAN-A1582 sequences: TtTAW1-1-1A and TtTAW1-1-1B.