*4.1. Plant Material and Standard Growth Conditions*

The generation of stable T4-generations of *Arabidopsis thaliana* (Arabidopsis) lines (ecotype Columbia 0, Col-0) overexpressing the photorespiratory phosphoglycolate phosphatase 1 (*PGLP1*, At5g36700, EC 3.1.3.18) was described previously [10]. Lines with an approximately 28% (O9) and 44% (O1) increase in PGLP activity compared to the wild type were used during this study. Prior plant cultivation, seeds of all genotypes were surface sterilized with chloric acid, sown on a soil (Type Mini Tray; Einheitserdewerk, Uetersen, Germany) and vermiculite mixture (4:1) and incubated at 4 ◦C for at least two days to break dormancy. Subsequently, plants were grown under environmental controlled conditions in growth cabinets (SANYO, Osaka, Japan; CLF Plant Climatics, Wertingen, Germany) with the following conditions as a standard: photoperiod - 10/14 h day/night-cycle, temperature - 20/20 ◦C day/night-cycle, photon flux density of ~120 μmol m−<sup>2</sup> s<sup>−</sup>1, 70% relative humidity, 0.039% CO2 in air (Figure 1A). During growth, plants were regularly watered with 0.2% Wuxal liquid fertilizer (Aglukon, Düsseldorf, Germany).

## *4.2. Stress Conditions*

Wild-type and *PGLP* overexpression plants were grown under standard conditions (Figure 1A), following exposure to two different stress conditions. First, simulating water-limiting conditions, all genotypes were grown conjointly in one pot (28 cm in diameter, 1 plant per genotype, 5 technical replicates) for 6 weeks with regular water supply to allow for a high level of comparability. After control experiments were carried out, watering was stopped, and experiments performed at intervals specified in the manuscript text. Second, simulating temperature stress, all genotypes were grown under standard conditions for 6 weeks and control experiments carried out. Subsequently, all plants were exposed to elevated temperatures (30 ◦C) with otherwise equal conditions. Photosynthetic measurements were performed, and leaf-material harvested after 1, 3 and 7 days in 30 ◦C.

## *4.3. qRT-PCR Analysis and Immunological Studies*

To follow the expression of selected photorespiratory genes and proteins we harvested leaf-material at the end of the day (9 h illumination) during temperature transition under control conditions (20 ◦C) and after 1, 3 and 7 days in 30 ◦C. For gene expression analysis total leaf RNA was extracted from ~100 mg tissue (pooled from three biological individuals) and ~2.5 μg used to synthesize cDNA (Nucleospin RNA plant kit, Macherey-Nagel; RevertAid cDNA synthesis kit, MBI Fermentas). Prior to qRT-PCR analysis, cDNA amounts were calibrated by RT-PCR according to signals from 432-bp fragments of the constitutively expressed 40S ribosomal protein *S16* gene, with oligonucleotides P444 [5 -GGC GAC ACA ACC AGC TAC TGA-3 ] and P445 [5 -CGG TAA CTC TTC TGG TAA CGA-3 ]. Detection and

normalization of gene expression were performed as described previously (Timm et al., 2013), and mRNA amounts of *PGLP1* (P393 [5 -CAG AAT GGC GGT TGT AAG AC-3 ] and P394 [5 -GGC TCC CTA ATT TGC TAT GC-3 ]; 328 bp), *GGT1* (P405 [5 -CGT TGC TCA GGC TCG TTC TC-3 ] and P406 [5 -CCA CCT CGC TGT CCA CAT TC-3 ]; 336 bp), *GDC-P1* (P366 [5 -AGC AAA TCC GTA GCC ATC AC-3 ] and P413 [5 -TAT GTC CAA TGC GTC GCT TC-3 ]; 327 bp), *GDC-T* (P367 [5 -GCA ATC AAT AAC CCG TCG TC-3 ] and P368 [5 -TCA ATG GCA CCT CCT TTC TC-3 ]; 363 bp), *SHM1* (P395 [5 -GCC CAG TGA AGC TGT TGA TG-3 ] and P396 [5 -AGT TGG CAG GAG ATC CAG AC-3 ]; 365 bp) and *HPR1* (P397 [5 -GGC TGA ACT AGC TGC TTC TC-3 ] and P398 [5 -GCA CCG GGT GAA GAC TTA TC-3 ]; 360 bp) quantified accordingly using the oligonucleotide combinations given in brackets after each gene. Abundances of selected photorespiratory proteins were analyzed by immunoblotting. Briefly, total leaf proteins were extracted from ~100 mg leaf tissue (pooled from three biological individuals) and 10 μg separated by SDS-PAGE followed by immunoblotting according to standard protocols. Alterations in protein expression were visualized using specific antibodies against PGLP1, GDC-P, SHM1 and HPR1 [38].

#### *4.4. Determination of Starch and Metabolite Analysis*

Starch contents were measured enzymatically as described previously [39] from ~50 mg of leaf tissue harvested at EoD (9 h illumination) from at least four biological replicates per genotype. The soluble fraction of the extraction procedure was further subjected to gas chromatography (GC) analysis to quantify sucrose, glucose and fructose as described previously [40]. Amino acids and organic acids were essentially quantified on a high-performance liquid chromatograph mass spectrometer LCMS-8050 system (Shimadzu, Japan) as described recently [41] from 50 mg leaf-tissue harvested at the end of the day (9 h of illumination). The compounds were identified and quantified using the multiple reaction monitoring (MRM) values given in the LC-MS/MS method package and the LabSolutions software package (Shimadzu, Japan). Authentic standard substances (Merck, Germany) at varying concentrations were used for calibration and peak areas normalized to signals of the internal standard (2-(N-morpholino)-ethanesulfonic acid - MES).

#### *4.5. Gas Exchange and Chlorophyll a Fluorescence Measurements*

All gas exchange parameters were determined in a 6-h time period between 2 h after onset and 2 h prior offset of illumination on a Li-Cor-6400 gas exchange system (LI-COR, Lincoln, NE, USA) using fully expanded leaved from plants at growth stage 5.1 [33]. Prior the actual measurement, leaves were pre-adapted to the measuring chamber for at least 10 min. To determine net CO2 compensation points (Γ), *A*/*Ci* curves (400, 300, 200, 100, 50, 20, 0, 400 ppm CO2) were recorded with the following conditions: photon flux density = 1000 μmol m−<sup>2</sup> s<sup>−</sup>1, chamber temperature = 20 ◦C, flow rate = 300 μmol s−<sup>1</sup> and relative humidity = 60% to 70%. Fluorescence light response curves (PAR: 1600, 1200, 800, 600, 400, 200, 100, 50, 20 and 0 μmol m−<sup>2</sup> s<sup>−</sup>1) were measured at two different block temperatures (20 ◦C and 30 ◦C) with the following conditions: CO2 concentration = 400 ppm; flow rate = 300 μmol s−<sup>1</sup> and relative humidity = 60 to 70%. Relative rates of electron transport around PSII at a given light intensity (PAR) were assessed by the formula ETR = YPSII × PAR × 0.84 × 0.5. The factors are based on the assumptions that 84% of the incident quanta are absorbed by the leaf (factor 0.84) and that the transport of one electron by the two photosystems requires the absorption of two quanta (factor 0.5). YPSII (effective quantum yield of photosystem II) was calculated as described previously [42] using the following formula:

#### *4.6. Statistical Analysis*

If values were described to be significantly different from the control within the text, the differences have been determined due to the performance of the two tailed Student s *t*-test algorithm incorporated into Microsoft Excel 10.0 (Microsoft, Seattle, WA, USA).
