Gene expression profiling in Rosa roxburghii fruit and overexpressing RrGGP2 in tobacco and tomato indicates the key control point of AsA biosynthesis

Abstract

Rosa roxburghii Tratt. is an important commercial horticultural crop endemic to China, which is recognized for its extremely high content of L-ascorbic acid (AsA). To understand the mechanisms underlying AsA overproduction in fruit of R. roxburghii, content levels, accumulation rate, and the expression of genes putatively in the biosynthesis of AsA during fruit development have been characterized. The content of AsA increased with fruit weight during development, and AsA accumulation rate was found to be highest between 60 and 90 days after anthesis (DAA), with approximately 60% of the total amount being accumulated during this period. In vitro incubating analysis of 70DAA fruit flesh tissues confirmed that AsA was synthesized mainly via the L-galactose pathway although L-Gulono-1, 4-lactone was also an effective precursor elevating AsA biosynthesis. Furthermore, in transcript level, AsA content was significantly associated with GDP-L-galactose phosphorylase (RrGGP2) gene expression. Virus-induced RrGGP2 silencing reduced the AsA content in R. roxburghii fruit by 28.9%. Overexpressing RrGGP2 increased AsA content by 8-12-fold in tobacco leaves and 2.33-3.11-fold in tomato fruit, respectively, and it showed enhanced resistance to oxidative stress caused by paraquat in transformed tobacco. These results further justified the importance of RrGGP2 as a major control step to AsA biosynthesis in R. roxburghii fruit.

Keywords: GDP-L-galactose pyrophosphatase (GGP); L-ascorbic acid (AsA); Rosa roxburghii Tratt.; fruits; overexpression.

Figure 1

Changes of R. roxburghii fruits during the development. (A) Fruit phenotypic changes, (B) dynamic changes of single fruit weight, (C) DHA and AsA content and (D) the rate of AsA accumulation during the development of R. roxburghii fruit. Values are means of 4 replicates ± SD. AsA accumulation rate = average increase in AsA content every day, taking the median point of adjacent time as the time node. Different letters denote statistical significance.

Figure 2

Changes of AsA content during incubation of AsA biosynthetic candidate precursors. AsA content changes following incubation of fruit flesh at 70 DAA with candidate precursors of AsA biosynthesis, at a concentrations of 15 mM. (A, B) included 4 and 5 candidate precursors, respectively, and water was used as a control.

Figure 3

The relative expression of genes involved in AsA biosynthesis during fruit development. L-galactose pathway: (A-H) D-galacturonic acid pathway: (I, H); Myo-inositol pathway: (J, K); L-gulose pathway: (D, K). For each sample, transcript levels were normalized to UBQ; expression over time was determined relative to a designation of ‘1’ at 15 DAA. Values are means of at least 3 replicates ± SD.

Figure 4

Phylogenetic relationships, gene structures and conserved protein motifs of plant GGP genes. (A, B) The un-rooted NJ phylogenetic tree of plant GGP genes. (C) Conserved motif compositions and distribution of the plant GGP genes. (D) Domain analysis of plant GGP genes. (E) Exon–intron structure of plant GGP genes. Phylogenetic analysis of the GGP genes in Arabidopsis thaliana, Solanum lycopersicum, Fragaria vesca, Pyrus bretschneideri, Mangifera indica, Actinidia chinensis, Nicotiana tabacum, Malus pumila and Rosa roxburghii. The unrooted tree was generated by MEGA7 using the conserved amino acid sequences of the 3 kinds RrGGP proteins. GGP protein groups were distinguished by different colors. The conserved motifs were detected using MEME software and represented by colored boxes. The length of GGP proteins can be estimated using the scale at the bottom, and the conserved motifs were shown in Table S2.

Figure 5

Silencing of RrGGP2 gene in R. roxburghii fruits. Detection of TRV2 transcripts (A), RrGGP2 expression levels (B) and AsA content (C) after VIGS. P, untreated; CK, treated with TRV2; pTRV2-RrGGP2, treated with pTRV2- RrGGP2. ** Represents P < 0.01 compared with CK.

Figure 6

AsA content, oxidative resistance and growth status of tobacco overexpressing RrGGP2. (A) PCR analysis for the presence of the RrGGP2 gene in Basta-resistant tobacco transgenic lines by cloning primers. (B) PCR-Southern dot blot analysis of the transgenic tobacco plants. Dot blot hybridization analysis following PCR products of the transgene RrGGP2 with CaMV 35S promoter-specific primer and OCS PolyA-specific primer, where the hybridization was performed with a CaMV 35S-specific sequence as a probe. (C) RT-PCR analysis of total RNA from transgenic tobacco with RrGGP2-specific primers. (D) AsA content in the six transgenic lines and WT plants. Phenotypic difference (E) and chlorophyll content (F) of leaf discs after paraquat treatments for 30 hr under continuous light at 22 ± 2 °C. * Represents P < 0.05 compared with WT2. Rooting status after one week (G) and fresh weight and shoot lengths after ten weeks (H) of transgenic tobacco plants and control on rooting medium of 150 mM NaCl. *Represents P < 0.05 compared with WT, white represents fresh weight, and black represents shoot length. Lanes P= positive control pPFGC5941-RrGGP2, WT = untransformed control plants, L1–L6 = Basta-resistant T2 transgenic tobacco lines.

Figure 7

AsA content in tomato overexpressing RrGGP2. Transgenic lines (A) and confirmation of the presence of transgenes in the RrGGP2-overexpressing tomato lines by a PCR amplification (B) and qRT–PCR (C). P, positive control, WT, untransformed control plants, OX-3–OX-5, Transgenic tomato plants, ** Represents p<0.01. AsA and DHA content in red fruit (D) and mature leaves (E) of RrGGP2 transgenic tomato lines. **Represents P < 0.01 compared with WT, white represents DHA, and black represents AsA.