Molecular Characterization and Expression Profiling of Tomato GRF Transcription Factor Family Genes in Response to Abiotic Stresses and Phytohormones

Abstract

Growth regulating factors (GRFs) are plant-specific transcription factors that are involved in diverse biological and physiological processes, such as growth, development and stress and hormone responses. However, the roles of GRFs in vegetative and reproductive growth, development and stress responses in tomato (Solanum lycopersicum) have not been extensively explored. In this study, we characterized the 13 SlGRF genes. In silico analysis of protein motif organization, intron-exon distribution, and phylogenetic classification confirmed the presence of GRF proteins in tomato. The tissue-specific expression analysis revealed that most of the SlGRF genes were preferentially expressed in young and growing tissues such as flower buds and meristems, suggesting that SlGRFs are important during growth and development of these tissues. Some of the SlGRF genes were preferentially expressed in fruits at distinct developmental stages suggesting their involvement in fruit development and the ripening process. The strong and differential expression of different SlGRFs under NaCl, drought, heat, cold, abscisic acid (ABA), and jasmonic acid (JA) treatment, predict possible functions for these genes in stress responses in addition to their growth regulatory functions. Further, differential expression of SlGRF genes upon gibberellic acid (GA3) treatment indicates their probable function in flower development and stress responses through a gibberellic acid (GA)-mediated pathway. The results of this study provide a basis for further functional analysis and characterization of this important gene family in tomato.

Keywords: GRF gene; Solanum lycopersicum; abiotic stress; fruit development; organ-specific expression; phytohormone treatment.

Figure 1

Sequence alignment of SlGRF (Solanum lycopersicum GRF) proteins and GRF proteins from Arabidopsis and rice: (a) the QLQ and WRC domains are indicated by the red box; and (b) the TQL and FFD motifs are green underlined. Identical amino acids are indicated by black and the amino acids with >50% similarity is indicated by gray background.

Figure 1

Sequence alignment of SlGRF (Solanum lycopersicum GRF) proteins and GRF proteins from Arabidopsis and rice: (a) the QLQ and WRC domains are indicated by the red box; and (b) the TQL and FFD motifs are green underlined. Identical amino acids are indicated by black and the amino acids with >50% similarity is indicated by gray background.

Figure 2

Phylogenetic analysis of GRF proteins from tomato, potato (St, Solanum tuberosum is used instead of PGSC0003DMT), Arabidopsis (Arabidopsis thaliana GRF, AtGRF), rice (Oryza sativa GRF, OsGRF), maize (Zea mays GRF-ZmGRF) and Chinese cabbage (Brassica rapa GRF, BrGRF). The phylogenetic tree was established with entire protein sequences from the above plant species by the UPGMA (Unweighted Pair Group Method with Arithmetic mean) method following the pair-wise deletion method. The numbers on the branches indicate bootstrap support values from 1000 replications. The scale represents the units of the number of amino acid substitutions per site. The protein sequences used in the phylogenetic analysis are listed in Additional File 1 with their accession IDs.

Figure 3

Exon–intron distribution of SlGRF genes. Exons and introns are represented by green boxes and black lines, respectively.

Figure 4

Expression of SlGRF genes in different organs. Root (R), stem (St), meristem (M), leaves (L), seedling (Se), flower bud (FB), full blooming flower (FF), and fruits at six developmental stages (1 cm: 1 centimeter-sized fruit, IM: immature fruit, MG: mature green fruit, B: breaker, B3: three days after breaker, B7: seven days after breaker) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Relative gene expression levels are normalized to EF1a (Elongation factor 1a) values. Error bars represent standard deviations of the means of three independent replicates. Statistically significant variations of expression and mean values at different sampling points (ANOVA, p ≤ 0.01 for all 12 genes) are indicated with different letters.

Figure 4

Expression of SlGRF genes in different organs. Root (R), stem (St), meristem (M), leaves (L), seedling (Se), flower bud (FB), full blooming flower (FF), and fruits at six developmental stages (1 cm: 1 centimeter-sized fruit, IM: immature fruit, MG: mature green fruit, B: breaker, B3: three days after breaker, B7: seven days after breaker) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Relative gene expression levels are normalized to EF1a (Elongation factor 1a) values. Error bars represent standard deviations of the means of three independent replicates. Statistically significant variations of expression and mean values at different sampling points (ANOVA, p ≤ 0.01 for all 12 genes) are indicated with different letters.

Figure 5

Expression of SlGRF genes in response to abiotic stresses: (a) NaCl; (b) drought; (c) heat; and (d) cold, at 0–24 h. The error bars represent the standard error of the means of three independent replicates of qRT-PCR analysis. Different letters associated with each treatment indicate statistically significant difference at 5% level of significance, where the same letter indicates that the values did not differ significantly at p ≤ 0.05 according to Tukey’s pairwise comparison tests.

Figure 6

Expression of SlGRF genes in response to phytohormone treatments: (a) gibberellic acid (GA3); (b) abscisic acid (ABA); and (c) jasmonic acid (JA) treatment at 0–24 h. The error bars represent the standard error of the means of three independent replicates of qRT-PCR analysis. Different letters associated with each treatment indicate statistically significant difference at 5% level of significance, where the same letter indicates that the values did not differ significantly at p ≤ 0.05 according to Tukey’s pairwise comparison tests.