Loss of decreased‐rubisco phenotype between generations of wheat transformed with antisense and sense rbcS

Abstract

SummaryThe elite UK winter wheat cv. Riband was transformed with constructs containing rbcS in sense and antisense orientations driven by the maize ubiquitin promoter with a transformation efficiency of 1.2%. Of 77 primary transformants 31% of the sense‐rbcS transformed lines and 78% of the antisense‐rbcS transformed lines had decreased rubisco content compared to wild‐type and marker‐only controls, with decreases of up to 60%. However, in the T1 progeny which inherited the transgene, only 5% showed significantly decreased rubisco content and these effects were on the margins of significance. Five potential T2 homozygous lines from T1 parents which had transgene segregation consistent with a single locus were identified. There was no significant decrease in rubisco content relative to wild‐type in any of these lines (LSD of 8% for P= 0.05). Expression of antisense rbcS transgenes in two of these T2 lines was low but was increased following exposure of the plants to 37°C for 48 h. However this did not induce a significant decrease in rubisco protein content relative to controls. Southern analysis of two antisense lines showed that they had low copy number and 1–2 insertion events. In one of the two lines there was increased methylation of the ubiquitin intron in T2 samples compared to the TO primary transformant. Further work is required to establish whether methylation occurred in all the lines which lost the phenotype, and therefore the likelihood of this being the cause. The disappearance of the decreased rubisco‐content phenotype between generations may therefore be attributable to (1) greater activity of the ubiquitin promoter due to greater stress in the T0 generation plants and/or (2) increased methylation of the transgene promoter region between generations.

Increased Rubisco Content in Transgenic Rice Transformed with the ‘Sense’ rbcS Gene

“…The reason for no increase in Rubisco protein in these plants is not known, but it is possible that the promoter used did not work efficiently. Mitchell et al (2004) described that the strength of the maize ubquitin promoter was not sufficient in wheat mesophyll tissues. Although the use of the CaMV 35S promoter has been successful in the antisense rbcS tobacco (Rodermel et al 1988, Hudson et al 1992, its characteristics are constitutive and not always efficient in green tissues in rice (Tada et al 1991).…”

“…Valjakka et al (2000) and Sillanpa¨a¨et al (2005) introduced a sense rbcS fused with the CaMV 35S promoter into silver birch. Mitchell et al (2004) also transformed wheat with a sense rbcS under the control of the maize ubiquitin promoter. Getzoff et al (1998) produced transgenic Arabidopsis with a construct of the sense rbcS from pea under the control of the CaMV 35S promoter.…”

“…Mitchell et al (2004) transformed wheat (Triticum aestivum) with rbcS in sense and antisense orientations driven by the maize ubiquitin promoter, but did not find any overexpression of Rubisco. In the case with wheat investigated by Mitchell et al (2004), introduction of the sense rbcS gene often resulted in decreased Rubisco content by co-suppression.…”

Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation

“…Our results, however, suggested that none of the transgenic lines accumulated more Rubisco than the wild type (Fig. 9B), and in transgenic wheat (Triticum aestivum), Ubi-RBCS expression failed to yield an increased Rubisco amount (Mitchell et al, 2004). On the other hand, we did not initially screen transformants for overexpression; rather, we sought lines with single insertions that correctly expressed the transgenes.…”

Photosynthetic characteristics in genetically modified sense-RbcS silver birch lines

“…The nuclear transformation approach has been applied to evaluate the contribution of Rubisco in controlling photosynthesis and growth in annual and herbaceous plants, such as tobacco (Rodermel et al, 1988;Stitt et al, 1991;Masle et al, 1993;Sicher et al, 1994), wheat (Mitchell et al, 2004) and rice (Makino et al, 1997(Makino et al, , 2000Suzuki et al, 2007Suzuki et al, , 2009a. However, none of these efforts has resulted in the improved specificity of Rubisco to CO 2 .…”