Evolution of ALOG gene family suggests various roles in establishing plant architecture of Torenia fournieri

Abstract

Background: ALOG (Arabidopsis LSH1 and Oryza G1) family with a conserved domain widely exists in plants. A handful of ALOG members have been functionally characterized, suggesting their roles as key developmental regulators. However, the evolutionary scenario of this gene family during the diversification of plant species remains largely unclear.

Methods: Here, we isolated seven ALOG genes from Torenia fournieri and phylogenetically analyzed them with different ALOG members from representative plants in major taxonomic clades. We further examined their gene expression patterns by RT-PCR, and regarding the protein subcellular localization, we co-expressed the candidates with a nuclear marker. Finally, we explored the functional diversification of two ALOG members, TfALOG1 in euALOG1 and TfALOG2 in euALOG4 sub-clades by obtaining the transgenic T. fournieri plants.

Results: The ALOG gene family can be divided into different lineages, indicating that extensive duplication events occurred within eudicots, grasses and bryophytes, respectively. In T. fournieri, seven TfALOG genes from four sub-clades exhibit distinct expression patterns. TfALOG1-6 YFP-fused proteins were accumulated in the nuclear region, while TfALOG7-YFP was localized both in nuclear and cytoplasm, suggesting potentially functional diversification. In the 35S:TfALOG1 transgenic lines, normal development of petal epidermal cells was disrupted, accompanied with changes in the expression of MIXTA-like genes. In 35S:TfALOG2 transgenic lines, the leaf mesophyll cells development was abnormal, favoring functional differences between the two homologous proteins. Unfortunately, we failed to observe any phenotypical changes in the TfALOG1 knock-out mutants, which might be due to functional redundancy as the case in Arabidopsis.

Conclusion: Our results unraveled the evolutionary history of ALOG gene family, supporting the idea that changes occurred in the cis regulatory and/or nonconserved coding regions of ALOG genes may result in new functions during the establishment of plant architecture.

Keywords: ALOG family; Duplication; MIXTA-like; Phylogeny; Plant architecture; Torenia fournieri.

Fig. 1

Bayesian phylogram (left) and motif analysis (right) of ALOG genes in three plants lineages. Physcomitrella patens and Sphagnum fallax were selected as outer groups. The Bayesian posterior probability is located in each node and the accession number can be found in each sequence. Seven ALOG genes cloned from Torenia fournieri were presented in red. The motif diagrams were generated in MEME and different colors represent different motifs

Fig. 2

Bayesian phylogram of ALOG genes in eudicots. Physcomitrella patens and Sphagnum fallax were chosen as outer groups. The Bayesian posterior probability is located in each node and the accession number can be found in each sequence. Seven ALOG genes cloned from Torenia fournieri were presented in red

Fig. 3

Reverse transcription polymerase chain reaction analysis (RT-PCR) of TfALOG genes. Gene names are shown on the left sides; PCR cycles are shown on the right sides in each gene. β-actin (TfACT3) was used as an internal control. R, root; S, stem; L, leaf; VA, vegetative apexes; RA, reproductive apexes; F9, stage 9 flower bud; F10, stage 10 flower bud; F11, stage 11 flower bud

Fig. 4

Subcellular localization of TfALOG-YFP fused proteins. Signals from YFP, mCherry, bright field and merged channels are shown in each assay; EV-YFP is an empty vector as a control; nuclear marker ARF19IV-mCherry plasmids were co-transformed with different YFP constructs; scale bars: 10 μm

Fig. 5

Phenotypic analysis of wild type (WT), 35S:TfALOG1 and 35S:TfALOG2 transgenic plants. a Three rows represent the over-all plants, flowers (bars: 10 mm) and epidermal cells in petal lobes (bars: 20 μm), two independent lines 35S:TfALOG1 16# and 35S:TfALOG1 26# were used for analysis. b Two rows represent the over-all plants and leaves (bars: 10 mm), two independent lines, 35S:TfALOG2 2# and 35S:TfALOG2 3# were used for analysis; white arrows indicate developmental defects on leaves

Fig. 6

Relative expression of TfMIXTA genes in wild type (WT) and 35S:TfALOG1 transgenic plants. Relative expression of TfMIXTA genes in different plants was determined by qRT-PCR in the stage 10 flower buds. Error bars represent ±1 SD from three biological replicates; statistically significant differences were marked by asterisks with *: P < 0.05. ***: P < 0.001