*2.5. Minor Changes in the Amino and Organic Acid Contents during Temperature Stress*

Considering the observed changes among carbohydrates, we were interested to which extent *PGLP* overexpression could also affect amino and organic acid levels in response to increased growth temperature. To this end, we used the same leaf-material harvested at EoD (9 h illumination, 20 ◦C and after 1, 3 and 7 days in 30 ◦C) for metabolite analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Among the amino acid profiles (Figure 6A), we found three general accumulation patterns. Ten amino acids (group A: asparagine, glutamate, histidine, isoleucine, leucine, phenylalanine, serine, tryptophan, tyrosine and valine) increased in their abundance after the transfer to elevated temperature, with highest amounts after three days at 30 ◦C, but distinctly drop at day 7 (Figure 6A) to almost control levels. Six amino acids (group B: cysteine, glutamine, glycine, lysine, proline and threonine) were characterized by a gradual decrease over the course of the entire experiment. A third pattern, comprising three amino acids (group C: alanine, aspartate and methionine), showed an initial drop at day one at 30 ◦C and then a subsequent increase until the initial levels at 20 ◦C was reached (Figure 6A). In general, the observed pattern was similar in wild-type and overexpressor plants. However, glycine, an amino acid directly related to photorespiration, was found

in lower amounts under control conditions and after three and 7 days at 30 ◦C in both overexpressor lines (Figure 6A). In addition, many amino acids showed changed levels at day 7 at 30 ◦C. For example, almost all of group A amino acids were decreased in the overexpressors after 7 days of temperature stress (Figure 6A), whereas group B and C representatives displayed slight increases after 7 days. Proline, as an exception, was higher in both lines one day after the shift to elevated temperature. Concerning the organic acids, we found that citrate and malate followed the same accumulation kinetics as group B amino acids, at least in the wild type (Figure 6B). Both tend to decrease over time but were significantly increased in the transgenic lines one (except O9 malate) and three days after the shift to 30 ◦C (Figure 6B). Compared to that, GABA and succinate fluctuate like group A amino acids, without major change in the different genotypes.

**Figure 5.** Carbohydrate contents of wild-type Arabidopsis and *PGLP* overexpressors exposed to 30 ◦C. Wild-type and *PGLP* overexpressor (O9 and O1) plants were grown in standard conditions (20 ◦C) for 6 weeks following exposure to elevated temperature (30 ◦C). Leaf material was harvested at the end of the day (9 h illumination) to determine absolute (**A**) starch, (**B**) sucrose, (**C**) glucose and (**D**) fructose contents under control conditions and after 1, 3 and 7 days after the transfer to 30 ◦C using gas chromatography. 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).

**Figure 6.** Amino acids and organic acid of wild-type Arabidopsis and *PGLP* overexpressors exposed to 30 ◦C. Wild-type and *PGLP* overexpressor (O9 and O1) plants were grown in under standard conditions (20 ◦C) for 6 weeks following exposure to elevated temperature (30 ◦C). Leaf material was harvested at the end of the day (9 h illumination) to determine absolute (**A**) amino acid and (**B**) organic acids contents under control conditions and after 1, 3 and 7 days after the transfer to 30 ◦C via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Shown are mean values ± SD of absolute metabolite concentrations (nmol g−<sup>1</sup> FW) from at least 4 biological replicates per genotype (Col.0—green, solid line with circles, O9—black, dotted lines with squares and O1—black, dashed line with triangles). Asterisks indicate values statistically different from the respective wild-type time point as determined by Student's *t*-test (*p* < 0.05).
