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. 2016 Mar 4;11(3):e0151067.
doi: 10.1371/journal.pone.0151067. eCollection 2016.

The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures

Zhengkun Qiu  1 , Xiaoxuan Wang  1 , Jianchang Gao  1 , Yanmei Guo  1 , Zejun Huang  1 , Yongchen Du  1
Affiliations

The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures

Zhengkun Qiu et al. PLoS One. .

Abstract

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Map-based cloning of the ah locus.
(A) Phenotypes of the NIL seedlings. (B) Total anthocyanin content in hypocotyls and leaves of NIL-PH and NIL-GH plants. (C) The phenotype of 5-leaf-old NIL-PH and NIL-GH plants after growth in a phytotron under 16 h of light at 16°C/ 8 h of dark at 8°C for 20 days. (D) Coarse linkage map of the green locus on chromosome 9, and high-resolution linkage map of ah (E), with the number of recombinants between the molecular marker and ah indicated. (F) Annotation of the candidate region surrounding ah, with dark gray boxes indicating the putative genes predicted in ITAG2.40. (G) AH structure and the mutation site in FMTT271. The black boxes represent the coding sequences and lines between boxes represent introns. (H) Relative expression levels of AH in hypocotyls and leaves of NIL-PH and NIL-GH plants. A tomato ACTIN (Solyc03g078400) gene was used as the reference gene. The hypocotyls from 6-old day and the leaves from 5-leaf-day seedlings were used. (I) Phylogenetic tree of AH and other bHLH proteins from several plant species, constructed using the neighbor-joining method. The bHLH proteins and their respective GenBank accession numbers are as follows: petunia AN1, AAG25927; petunia JAF13, AAC39455; maize Lc, NP_001105339; Arabidopsis MYC1, NP_191957; tobacco AN1b, AEE99258; tobacco AN1a, AEE99257; Arabidopsis GL3, NP_680372; Arabidopsis EGL3, NP_176552; Arabidopsis TT8, CAC14865; maize IN1, AAB03841; Medicago TT8, XP_003590656; tobacco AN1-LIKE, NP_001289495; maize Hopi, CAB92300. NIL-PH (GH) refers to the plants from the NIL population with purple (green) in hypocotyls. Three biological replicates of all samples were analyzed. Scale bars, 1 cm.
Fig 2
Fig 2. Analyses of AH expression and anthocyanin content in leaves of control and AH over-expressing transgenic tomato lines.
(A-F) Phenotypes of hypocotyls, leaves and fruits of the control and AH-expressing lines. Scale bars, 1 cm. (G) Total anthocyanin content in leaves of the control and AH-expressing lines (T1-1 to T1-9). (H) Relative expression levels of AH in leaves of the control and AH-expressing lines (T1-1 to T1-9). T1 generation plants were used for the analyses. Data presented here are the means of three replicates with error bars indicating ±SD.
Fig 3
Fig 3. Developmental and low temperature-induced anthocyanin accumulation and expression of AH.
(A) The phenotype of NIL-PH seedlings at five stages (2-day old to 12-day old). The seedlings were growing under normal condition (28°C/20°C, day/night). Anthocyanin content and relative expression levels of AH (B) and anthocyanin biosynthetic genes (C and D) in hypocotyls at different developmental stages. Hypocotyls from 20 seedlings from the five stages, respectively, were used. Relative expression levels of AH (E) and anthocyanin content (F) in leaves grown under 16 h of light at 16°C/ 8 h of dark at 8°C or 16 h of light at 28°C/ 8 h of dark at 20°C conditions. 5-leaf old tomato seedlings were used. The X axis indicates the duration of the different temperature treatments. Data presented are the mean of three biological replicates with error bars indicating ±SD. Scale bars, 1 cm.
Fig 4
Fig 4. Identification of differentially expressed genes between NIL PH and GH hypocotyls.
Hypocotyls from 20 5-day old seedlings from NIL PH and GH seedlings were used. Two biological replicates of all samples were performed. (A) The relative expression pattern of PH and GH differentially expressed genes. (B) The enriched GO terms in biological processes of the up-regulated genes in PH compared with GH. (C) The enriched GO terms in biological processes of the down-regulated genes in PH compared with GH. (D) The relative expression pattern of anthocyanin biosynthetic genes in PH and GH. (E) The relative expression pattern of bHLH and MYB transcription factors (TFs) among the differentially expressed genes.
Fig 5
Fig 5. Identification of differentially expressed genes between NIL 16-PL and 16-GL seedlings under low temperature stress.
Five-leaf old NIL PH and GH seedlings grown at 28°C were treated at 28°C/20°C (day/night, 16h/8h, named 28-PL and 28-GL, respectively) or 16°C/8°C (day/night, 16h/8h, named 16-PL and 16-GL, respectively) for 5 days. RNA was extracted from leaves of individual lines and used for RNA-seq profiling. Two biological replicates of all samples were prepared. (A) The relative expression pattern of differentially expressed genes between 16-PL and 16-GL seedlings under low temperature conditions, excluding the differentially expressed genes from the 28-PL and 28-GL comparison. The genes in red color mean that the same expression pattern of these genes were observed in NIL-PH relative to NIL-GH. (B) The relative expression pattern of anthocyanin regulator genes AH, SlAN2, SlANT1, SlANT1-like and SlAN2-like in all samples. The correlation value of SlAN2, SlANT1-like to AH was evaluated by the normalized RPKM (log2(RPKM+0.001)) from the 6 samples, respectively.
Fig 6
Fig 6. Phenotypes of anthocyanin deficient and anthocyanin enriched plants under cold stress.
Five-leaf old plants were treated at 4°C for 0 days and 3 days. (A) H2O2 accumulation in the leaves of the anthocyanin deficient plants, FMTT271 and NIL-GH, and the anthocyanin-enriched plants, NIL-PL and p35S-AH-FMTT271, as determined by DAB staining. (B) Cold-induced cell death in the mutant, FMTT271, NIL-GH and wild-type NIL-PL and p35S-AH-FMTT271 plants. Detached leaves were stained with trypan blue. Images are of representative plants. Scale bars, 0.2 cm.
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