Two LEAFY homologs ILFY1 and ILFY2 control reproductive and vegetative developments in Isoetes L

Abstract

LEAFY (LFY) is a plant-specific transcription factor, which is found in algae and all land plants. LFY homologs exert ancestral roles in regulating cell division and obtain novel functions to control floral identity. Isoetes L. is an ancient genus of heterosporous lycophytes. However, characters about LFY homologs in lycophytes remain poorly investigated. In this study, two LFY homologs, ILFY1 and ILFY2, were cloned from five Isoetes species, including I. hypsophila, I. yunguiensis, I. sinensis, I. orientalis, and I. taiwanensis. The full length of ILFY1 was 1449-1456 bp with an open reading frame (ORF) of 927-936 bp. The full length of ILFY2 was 1768 bp with ORF of 726 bp. Phylogenetic tree revealed that ILFY1 and ILFY2 were separated into two clades, and I. hypsophila were separated with the others. Expression analysis demonstrated that IsLFY1 and IsLFY2 for I. sinensis did not show functional diversity. The two transcripts were similarly accumulated in both vegetative and reproductive tissues and highly expressed in juvenile tissues. In addition, the IsLFY1 and IsLFY2 transgenic Arabidopsis similarly did not promote precocious flowering, and they were inactive to rescue lfy mutants. The results facilitate general understandings about the characteristics of LFY in Isoetes and evolutionary process.

Figure 1

Gene structures of ILFY1 and ILFY2 homologs. Three exons and two introns were shown as green boxes and black lines, respectively. The ILFY1 and ILFY2 sequences were identical for I. yunguiensis, I. sinensis, I. orientalis, and I. taiwanensis. The two introns were variable in the five species.

Figure 2

Sequences comparison of deduced LFY proteins. Deduced amino acid sequences of IhLFY1 and IhLFY2 for I. hypsophila, as well as IsLFY1 and IsLFY2 for I. sinensis were compared with LFY homologs, including LFY (Arabidopsis thaliana, AAA32826), FLO (Antirrhinum majus, AAA62574.1), PRFLL (Pinus radiata, O04116), NEEDLY (Pinus radiata, AAB68601.1), CRLFY2 (Ceratopteris richardii, BAB41070.2), SmLFY1 (Selaginella moellendorffii, XP_002978027), PpLFY1 (Physcomitrella patens, BAB60676.1), and PpLFY2 (Physcomitrella patens, BAB60677.1). Conservatively substituted and identical residues were depicted on a red background, and slightly conserved residues were on an orange background. N-terminal and C-terminal domains were overlined in blue and red, respectively. Second structures of the C-terminal domains including two β-sheet and seven α-helix, were indicated. Gaps were shown as dashes to maximize the alignments. The blue triangle represented a conspicuously different amino acid between P. patens and the other species.

Figure 3

Phylogenetic tree of LFY homologs. The phylogenetic tree was constructed using the amino acid sequences of LFY homologs. Numbers above the branches represent bootstrap value, and bootstrap less than 50% was removed. Accession numbers for the LFY homologs in the dataset were listed in Table S5.

Figure 4

Expression patterns of IsLFY1 and IsLFY2 using quantitative real-time PCR assays. The abbreviations L, Me, Mi, R, Co represent leaves, megasporangia, microsporangia, roots, and corms, respectively. The megasporangia were in the bottom of fertile leaves from 1th to 7th whorls, and microsporangia were from 8th to 11th whorls. The immature leaves were in the 12th whorl. Values represent the means ± SE. Asterisk indicated that the expression levels of IsLFY1 and IsLFY2 were significantly different with the P value less than 0.01.

Figure 5

In situ hybridization of IsLFY1 expressions. (a) Transverse section of roots. (b) Transverse section of leaves. (c) Transverse section of corms. (d) Longitudinal section of corms. (e) Transverse section of microsporangia. (f) Transverse section of megasporangia. Scale bars were 300 μm for megasporangia and microsporangia, 200 μm for root, leaves, and 100 μm for corms.

Figure 6

In situ hybridization of IsLFY2 expressions. (a) Transverse section of roots. (b) Transverse section of leaves. (c) Transverse section of corms. (d) Longitudinal section of corms. (e) Transverse section of microsporangia. (f) Transverse section of megasporangia. Scale bars were 300 μm for megasporangia and microsporangia, 200 μm for root, leaves, and 100 μm for corms.

Figure 7

Phenotypic characters in Arabidopsis of constitutive IsLFY1 and IsLFY2 expressions. Figures from a to d are related to transgenes that the genes were crossed into wild Arabidopsis plants. Four-week IsLFY1 transgenic line (a), wild Arabidopsis plants (b), and IsLFY2 transgenic line (c). The IsLFY1 and IsLFY2 transgenic plants did not differ significantly in appearance from the wild-type plant. Flowers for the transgenic plants were consistent with the wild plants (d). Figures from e to j are related to transgenes that the genes were crossed into lfy-1 mutants. lfy-1 mutant (e). IsLFY1 transgenic line (f). IsLFY2 transgenic line (g). The IsLFY1 and IsLFY2 were inactive to rescue the lfy mutants. The transgenic plants and lfy mutant consistently showed that the early flowers were replaced by bracts, which subtended secondary inflorescences (h). The later flowers consisted of sepals and carpels (i and j).