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. 2018 Mar 28;7(2):27.
doi: 10.3390/plants7020027.

Identification of Leaf Promoters for Use in Transgenic Wheat

Saqer S Alotaibi  1   2 , Caroline A Sparks  3 , Martin A J Parry  4   5 , Andrew J Simkin  6   7 , Christine A Raines  8
Affiliations

Identification of Leaf Promoters for Use in Transgenic Wheat

Saqer S Alotaibi et al. Plants (Basel). .

Abstract

Wheat yields have plateaued in recent years and given the growing global population there is a pressing need to develop higher yielding varieties to meet future demand. Genetic manipulation of photosynthesis in elite wheat varieties offers the opportunity to significantly increase yields. However, the absence of a well-defined molecular tool-box of promoters to manipulate leaf processes in wheat hinders advancements in this area. Two promoters, one driving the expression of sedoheptulose-1,7-bisphosphatase (SBPase) and the other fructose-1,6-bisphosphate aldolase (FBPA) from Brachypodium distachyon were identified and cloned into a vector in front of the GUS reporter gene. Both promoters were shown to be functionally active in wheat in both transient assays and in stably transformed wheat plants. Analysis of the stable transformants of wheat (cv. Cadenza) showed that both promoters controlled gus expression throughout leaf development as well as in other green tissues. The availability of these promoters provides new tools for the expression of genes in transgenic wheat leaves and also paves the way for multigene manipulation of photosynthesis to improve yields.

Keywords: photosynthesis; promoter; reporter gene; tissue specific; wheat; yield.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of a two-kilobase upstream region of the sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphate (FBPA) genes from B. distachyon showing potential regulatory motifs. ATG denotes the codon initiating translation of the SBPase and FBPA proteins.
Figure 2
Figure 2
Histochemical GUS assay of agroinfiltrated N. benthamiana leaves. The transient expression assays were performed on four- to five-week old N. benthamiana leaves, incubated at 24 °C in 16 h/8 h light/dark for 72 h after infiltration prior to GUS staining: (A) gus expression driven by the SBPase promoter; (B) gus expression driven by the FBPA promoter; and (C) control (wild type: non-infiltrated leaf).
Figure 3
Figure 3
Histochemical GUS assay for transient expression of B. distachyon SBPase and FBPA promoters in wheat leaves. The transient assays were performed by particle bombardment of young wheat leaves which were incubated at 22 °C in 12 h/12 h light/dark for 48 h prior to GUS staining: (A) gus expression driven by the SBPase promoter; (B) gus expression driven by the FBPA promoter; (C) control leaf bombarded without DNA.
Figure 4
Figure 4
Histochemical analysis of GUS activity in T1 wheat lines stably transformed with the B. distachyon SBPase or FBPA promoter constructs. gus expression driven by (A) SBPase promoter and (B) FBPA promoter in tissue from young seedlings, from flag leaves of mature plants and in flowers and roots. WT = wild type infiltrated tissue is used as a control. (C) Microscopic observation of localization of gus expression in T1 wheat leaves of two independent lines for each construct compared to plants transformed with the bar (Nos:Basta) selectable marker gene construct only (pRRes1.111).
Figure 5
Figure 5
Histochemical analysis of GUS activity in T2 wheat lines stably transformed with the B. distachyon SBPase or FBPA promoter constructs. Leaves were taken for GUS staining at three different growth stages (seedling, elongation and flag leaf) together with samples from different tissues: flowers and roots. Three different lines per promoter were analyzed and compared to WT (wild type) and CN (control plants transformed with bar gene only).
Figure 6
Figure 6
Analysis of transcript expression of the codon optimized P. umbilicalis cytochrome c6 [12]) with a FLAG tag (CytC6-FLAG) gene driven by the B. distachyon (A) SBPase or (B) FBPA promoters in stably transformed wheat T0 lines. Relative expression levels and range of expression in these 30 independent transgenic plants compared to the expression of the endogenous SBPase in the same plants. Data are presented as a % of the endogenous SBPase expression and standard error of nine technical reps are shown. (C) FLAG-tagged protein accumulation in a selection of FBPA promoter wheat lines.
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