## 4 Discussion
On the contrary, the decline in enzyme activity observed in the NT plants was probably due to the adverse effects of drought on the overall plant metabolism (Figure 2), supporting the claims that an increased trehalose synthesis in plants aids in better performance under abiotic stress conditions [10; 32]. Despite being transformed with the same promoter, lines rd-1B and rd-1C showed some differences in the response against drought. The differences could be due to diverse insertion sites of the transgene. Plant transformation using _Agrobacteriumium_ is random rather than site-directed; thus, the actual transgene insertion site may influence gene expression. Nonetheless, the overall effect in the two lines transformed with the rd29A promoter was similar in both lines: because there was an improved tolerance to drought compared to the NT plants. The sucrose and trehalose content (Table 2) and enzyme activity data in the 35S lines (Figure 3) suggest a split between trehalose and sucrose synthesis, as both sugars are synthesized in the cytoplasm. In the 35S line, this could also explain the lower RuBP regeneration shown by the low FBPase activity (Figure 2b), indicating that the constitutive overexpression of the TPSTP enzyme in wheat directed the flux of triose phosphates towards the cytoplasm rather than to the chloroplast. Seeds from the 35S line had the lowest germination rate compared to the lines transformed with the rd29A promoter and NT seeds. Moreover, during the development of the 35S plants, the leaves were narrow (Figure S1). The changes in the leaves were probably caused by a reduced starch accumulation in the chloroplast during photosynthesis. On the one hand, in lines rd-1B and rd-1C, trehalose increased once the soil water diminished (Table 2), which corresponds with the induction of the TPS-TPTP bifunctional enzyme by the rd29A promoter under drought stress. On the other hand, the sucrose content in the rd-1B and rd-1C lines diminished when drought increased (Table 2), coinciding with the low SPS activity (Figure 3c). These results suggest that triose phosphates were mainly utilized for trehalose synthesis and RuBP regeneration in these lines when drought was imposed, which helped the transgenic plants to maintain better photosynthesis during stress. Furthermore, because rd-1B and rd-1C had a 100% germination rate and produced more and broader leaves, the increased CO2 fixation and triose phosphate flux in the chloroplast likely allowed a higher starch accumulation in these transgenic lines. The overexpression of _TPS_ and/or _TPP_ genes in plants seems to have different effects on the trehalose content depending on the species; some results even seem contradictory. [PERSON] et al. (2000) and [PERSON] et al. (2007) [19; 22] found increased trehalose content without correlation with gene expression levels. [PERSON] et al. (2003) [16] reported high trehalose accumulation and high expression of the _TPS-TPP_ gene from _E. coli_. In tobacco plants, the overexpression of a yeast TPS1 accumulated less trehalose than the non-transformed plants despite increased TPS activity, and no effect in the trehalose content was found in the _E. coli_ TPS and TPP [17; 23] overexpressed plants. No previous studies on wheat transformed for TPS and/or TPP overexpression have been published so far. In plants, trehalose accumulation is only known in resurrection plants. The lack of trehalose accumulation in higher plants is thought to be due to the increased activity of trehalose [38]. Here, a high content of trehalose from day 2 to day 8 of drought in the NT plants was detected (Table 2). Moreover, the trehalose activity was higher in the transgenic lines than in the NT plants under drought (0.03 nmol min\({}^{-1}\) mg\({}^{-1}\) in NT versus 0.2-0.5 nmol min\({}^{-1}\) mg\({}^{-1}\) in transgenic plants), and this explains the lower trehalose content in the transformed plants. Similar results were reported for transgenic tobacco and potato plants, with no increase in trehalose but enhanced stress tolerance [17; 39].