*2.3. High PGLP Activity is Beneficial for Photosynthesis at Increased Growth Temperatures*

Next, we aimed to know whether improved maintenance of photosynthesis was restricted to water-limiting conditions (Figure 3B) or if it also occurs in response to other abiotic stresses in *PGLP* overexpressor lines. Therefore, we tested increased growth temperature, which is anticipated to stimulate 2-PG production. To this end, the transgenic lines and the wild type were grown for 6 weeks under standard conditions and then exposed to elevated temperatures (30 ◦C; Figure 1D). As proxies for their capability to acclimate to higher temperatures, we characterized photosynthetic parameters such as *A* and Γ under control conditions and after one and 7 days at 30 ◦C. As before, we did not measure significant differences between the genotypes under control conditions. As expected, transfer of the plants to 30 ◦C decreased *A* and increased Γ. *A* decreased to the same extends in the transgenic lines and the wild type (Figure 4A). However, after 7 days exposure to 30 ◦C, Γ is significantly decreased in both transgenic lines, reaching almost control levels, while it remained at a similarly high level in wild type as found after only one day at 30 ◦C (Figure 4B, significant in O1). This change in line O1 was accompanied by significantly increased *gs* (Col.0—0.1032 ± 0.0202 versus O1—0.1985 ± 0.0726\*) and *E* (Col.0—1.67 ± 0.28 versus O1—2.96 ± 0.86\*), which remained similar between O9 and the wild type (*gs*—0.1266 ± 0.0250; *E*—1.92 ± 0.33). Collectively, O1, the line with lowest 2-PG contents [10], has a significant advantage over the wild type after exposure to elevated growth temperatures for one week. This beneficial effect is likely not due to enhanced CO2 fixation, but rather due to improved carbon utilization as suggested by the lower Γ.

**Figure 4.** Photosynthetic parameters of wild-type Arabidopsis and *PGLP* overexpressors exposed to 30 ◦C. Wild-type and *PGLP* overexpressor (O9 and O1) plants were grown under standard conditions (20 ◦C) for 6 weeks following exposure to elevated temperature (30 ◦C). Gas exchange measurements were carried out to determine (**A**) CO2 assimilation rates (*A*) and (**B**) net CO2 compensation points (Γ) under control conditions (20 ◦C) and after 1 and 7 days after the transfer to 30 ◦C. Shown are mean values ± SD from at least 4 biological replicates per genotype. Asterisks indicate values statistically different from the wild type as determined by Student's *t*-test (*p* < 0.05; n. s.—not significant).

#### *2.4. Improved 2-PG Degradation Translates to Higher Transitory Starch Stocks under Temperature Stress*

Given that 2-PG levels are inversely correlated with starch accumulation [10], we quantified the amounts of starch and soluble sugars at the end of the day (EoD) in leaf-material from the temperature shift experiment. In agreement with our previous report [10], the overexpressors O9 and O1 contained significantly elevated starch contents (~27%) at EoD under control conditions compared to the wild type (Figure 5A). After the transfer to 30 ◦C, wild-type starch levels significantly drop compared to the control, which is in agreement with other reports [34]. Notably, both transgenic lines were able to maintain higher starch accumulation after the temperature increase, which was clearly pronounced 7 days after the shift to 30 ◦C. At this time point, O9 starch increased to about 46% and O1 to about 123% (Figure 5A). About the contents of soluble sugars, we did not find significant change in sucrose (Figure 5B). Glucose was similar in all plants at 20 ◦C. However, it significantly decreases in both lines after one to three days at 30 ◦C and increased again after 7 days at 30 ◦C (Figure 5C). Changes in fructose were only seen after one day at 30 ◦C, when both overexpressor lines displayed a significant drop compared to wild type (Figure 5D). Collectively, our results suggest that faster 2-PG removal and sustained photosynthesis are beneficial for carbon allocation towards transitory starch under control as well as stress conditions, without impacting steady-state sucrose amounts.
