Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses

Abstract

Background: Citrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported.

Results: Here, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress.

Conclusions: Taken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.

Keywords: Citrus sinensis; Expression pattern; Functional identification; GRF transcription factor; Genome-wide identification.

Fig. 1

Phylogenetic analysis of GRFs in Citrus sinensis (Cs), Arabidopsis thaliana (At), Oryza sativa subsp. japonica (Os), Populus trichocarpa (Ptr), Pyrus bretschneideri (Pb) and Vitis vinifera (Vv). The phylogenetic tree was created using MEGA X by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. The species background for each GRF protein is represented by different colors. Based on the bootstrap values and evolutionary distances, the tree was clustered into five subfamilies (I-V)

Fig. 2

Conserved motif and gene structure analysis of CsGRFs. A Phylogenetic relationship of CsGRF proteins. B The distribution of 6 conserved motifs in CsGRF proteins, identified by MEME program, was shown by different colored blocks. The sequences of these conserved motifs were listed in Supplementary Table S3. C Exon/intron structures of CsGRFs. The exons and introns were represented by pink boxes and black lines, respectively. The blue boxes indicated the upstream and/or downstream untranslated region

Fig. 3

Duplication and synteny analyses of GRF genes among Citrus sinensis, Arabidopsis thaliana and Oryza sativa. A Location and the collinearity analysis of CsGRFs. The green columns represent chromosomes with the chromosome numbers placed in the middle and the gene ID shown outside the plot. The blue line inside the plot indicated the the genes located on the duplicated segmental regions between CsGRFs. B Collinearity relationship of GRF genes among Citrus sinensis, Arabidopsis thaliana and Oryza sativa. The horizontal columns represent chromosomes with the chromosome numbers placed in the middle. The gray lines indicated the collinear blocks within each two genome pairs, and the identified syntenic CsGRF genes are linked by red lines

Fig. 4

cis-element analysis in the promoters of CsGRFs. cis-regulatory stress-responsive elements were identified in the 2.5 kb upstream promoter region of CsGRFs. Different colored rectangles represent different elements. Detailed information of sequence and position of these elements was described in Supplementary Table S6

Fig. 5

Expression profiles of CsGRFs under multiple phytohormone treatments. Expression analysis was carried out in leaves of C. sinensis at different time points (0 h, 3 h, 6 h, 12 h, 24 h and 48 h after treatments). The qPCR results of CsGRFs were normalized by log₂ transform. The heatmap constructed by TBtools software. Color scale erected horizontally at the bottom of the diagram

Fig. 6

Expression profiles of CsGRFs under multiple abiotic stresses. Expression analysis was carried out in leaves of C. sinensis at different time points (0 h, 3 h, 6 h, 12 h, 24 h and 48 h after NaCl and cold treatments, 0 h, 0.5 h, 1 h, 3 h, 6 h and 12 h after dehydration treatment). The qPCR results of CsGRFs were normalized by log₂ transform. The heatmap constructed by TBtools software. Color scale erected horizontally at the bottom of the diagram

Fig. 7

Venn diagram of CsGRFs under phytohormone treatments and abiotic stresses. A Diagram of overlapping CsGRFs which showed up-regulated expression levels under phytohormone treatments and abiotic stresses. The red columns represent the number of overlapping treatments with up-regulated expression pattern under phytohormone treatments and abiotic stresses. The black columns in the lower left corner represent the number of up-regulated CsGRFs under each treatments. The black circles strung with lines represent the overlapping treatments. B Diagram of overlapping CsGRFs which showed down-regulated expression levels under phytohormone treatments and abiotic stresses. The green columns represent the number of overlapping treatments with down-regulated expression pattern under phytohormone treatments and abiotic stresses. The black columns in the lower left corner represent the number of down-regulated CsGRFs under each treatments. The black circles strung with lines represent the overlapping treatments

Fig. 8

Identification, expression analysis and phenotypic observation of CsGRF04-VIGS transgenic plants. A Genomic PCR for identification of the CsGRF04-VIGS plants. Full-length gels are presented in Supplementary Figure S2. B Expression of CsGRF04 in ten randamly selected positive VIGS lines, as analyzed by qPCR. C Phenotypic observation of WT and CsGRF04-VIGS plants. D, E Length (D) and width (E) statistics of leave from WT and CsGRF04-VIGS plants. The asterisk indicates the significant difference between WT and the CsGRF04-VIGS plants based on a Tukey’s test (*** p < 0.001). The scale bar indicates 1 cm

Fig. 9

Silencing of CsGRF04 by virus-induced gene silencing (VIGS) alters abiotic stress resistance in C. sinensis. A Phenotype of 1-month-old WT and CsGRF04-VIGS plants before (left panels) and after (right panels) salt treatment. NC: normal condition. B, C Electrolyte leakage (EL) (B) and chlorophyll content (C) of WT and CsGRF04-VIGS plants before and after the salt treatment. D Phenotype of 1-month-old WT and CsGRF04-VIGS plants under NC (left panels) and after recovery (right panels) of cold treatment (8 h at -4 °C and 3 days at ambient temperature). E, F EL (E) and chlorophyll content (F) of WT and CsGRF04-VIGS plants before and after the cold treatment. G Phenotype of 1-month-old WT and CsGRF04-VIGS plants before (left panels) and after (right panels) drought treatment. H, I EL (H) and chlorophyll content (I) of WT and CsGRF04-VIGS plants before and after the drought treatment. Error bars represent ± SE (n = 3). ns: not significant. Asterisks indicate significant differences between CsGRF04-VIGS and WT plants (***P < 0.001). The scale bar indicates 1 cm