Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology

Abstract

Background: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development.

Results: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity.

Conclusions: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

Keywords: Citrus (Citrus sinensis); Fruit-specific expression; Plum (Prunus americana); Promoter; Tomato (Solanum lycopersicum); Transgenic.

Fig. 1

Candidate citrus gene expression patterns in different tissues. A heatmap displaying publicly available citrus gene expression data for four selected candidate genes is shown. The normalized expression levels are colored according to the scale bar shown on the top of the image. Each column represents a single biological replicate of the indicated sample, each row represents the candidate gene. The four candidate citrus genes were selected based on their high-level of expression in fruits and flowers compared to low or undetectable expression in leaves

Fig. 2

Schematic representation of the promoter-GUSPlus construct T-DNA. The T-DNA of the pCTAGII-GUSPlus binary vector is shown. It harbors a codA-nptII fusion gene selection marker under the control of the Arabidopsis Ubiquitin10 promoter (Ubi10p) and nopaline synthase terminator (T). Candidate fruit-specific promoters from citrus (CitSEPp, CitWAXp, CitUNKp, CitJuSacp, CitVO1) and plum (PamMybAp), as well as control fruit-specific promoters from tomato (E8p and PGp) are shown. The T-DNA also contains attP and res recombination recognition sites for the Bxb1 and CinH recombinase enzymes respectively. LB and RB designate the Agrobacterium left and right borders

Fig. 3

Agroinjection-mediated transient expression in tomato fruit. Results from an Agrobacterium-mediated transient expression assay testing the functionality of the candidate fruit-specific promoters in unripe (left) and ripe (right) tomato fruits is shown. Control, wildtype tomato fruit infiltrated with an empty vector is shown on the top, followed below by those infiltrated with the promoter construct as labeled. Each fruit was histochemically stained for β-glucuronidase activity 4 days after agroinjection. The CitVO2p construct is a CitVO1 promoter fragment lacking the native intron. Scale bar – 1 cm

Fig. 4

Histochemical staining of vegetative and fruit tissues from wildtype and transgenic tomato lines. Histochemical staining of whole seedlings mature leaves, flowers, cross sections of unripe and ripe fruit, and whole unripe fruit are shown. Each row contains representative images from wildtype or a corresponding promoter-GUSPlus transgenic line. Scale bar – 1 cm

Fig. 5

Seedless phenotype in transgenic CitJuSacp-GUSPlus tomato lines. a Wildtype and b CitJuSacp transgenic green and ripe fruit. c The measured genomic DNA content (picograms/2C) in CitJuSacp (JS), CitWAXp (WAX) and CitUNKp (UNK) transgenic lines and Micro-Tom wildtype (WT) lines. Scale bar =1 cm

Fig. 6

β-glucuronidase activity in measured in tomato fruit and leaves. The measured β-glucuronidase activity in leaves (green bars), unripe fruit (yellow bars) and ripe fruit (red bars) from wildtype (WT) and representative transgenic tomato lines is shown. The mean of nine samples (except CsJuSacp with n = 6), with their standard error bars (the error bars) are shown