Salicylhydroxamic acid

Total Chl, Chla and Chlb content was determined according to Lichtenthaler (1987 (link)). Fe content in purified mitochondria was determined by ICP-MS spectroscopy (Variant).
F₀F₁ATP synthase and G6PDH activities were performed according to Camacho-Pereira et al. (2009 (link)).
O₂ consumption and use of KCN and SHAM (salicylhydroxamic acid) was measured on root apices from 10 days-old plants according to Vigani et al. (2009 (link)).
Protein sequence alignment was performed with Multalin version 5.4.1 (Corpet, 1988 (link)) at http://multalin.toulouse.inra.fr/multalin/.

Respiration was measured on the basis of the method described by Wang et al. [43 (link)]. First, 0.03 g roots were cut into 2 mm small segments and put into 2 mL 50 mM phosphate buffer (pH 6.8). After reaction for 2 min at room temperature, the oxygen value slope was defined as the total respiration rate (V_t). Then, 2 mM KCN or 2 mM salicylhydroxamic acid (SHAM) was added and reacted for 2 min; the oxygen value slope was defined as the AP capacity (V_alt) or the CP capacity (V_cyt), independently.

The crystalline sample of SHA (98%, Sigma Aldrich, Merck, KGaA, Darmstadt, Germany) was allowed to sublimate at 410 K from a small electric oven assembled inside the vacuum chamber of the cryostat. The temperature of the oven was controlled by the DC-regulated power supply (NDN instruments). The matrices were obtained by the co-deposition of salicylhydroxamic acid vapor with a large excess of argon (nitrogen) into the cold CsI window. The matrix concentration was controlled by the matrix gas flow rate, which was adjusted to minimize the concentration of SHA aggregates and thermolysis products. The low temperature was maintained by means of a closed-cycle helium refrigerator (ARS-2HW, APD-Cryogenics). The FTIR spectra were recorded between 4000 and 500 cm⁻¹ in a transmission mode by means of a Nicolet iS50 FTIR spectrometer with a resolution of 0.5 cm⁻¹, using a liquid N₂-cooled MCT detector. Photochemical reactions were induced in the SHA/Ar (N₂) matrices by UV radiation of a pulsed (7 ns) optical parametric oscillator Vibrant 355 (Opotek, Inc., Carlsbad, CA, USA) (repetition rate 10 Hz, average pulse energies ~7.0 mJ (325 nm) and ~2.5 mJ (250 nm)), pumped with a pulsed Nd:YAG laser (Quantel, Bozeman, MT, USA). The experiments started using λ = 400 nm light and proceeded with a gradual decrease in the output wavelength. The process was controlled by recording the infrared spectra of the matrix after each irradiation. The best experimental conditions for the observation of SHA photochemistry were obtained by irradiation with a wavelength of λ = 340 nm.
All the calculations were performed with the Gaussian 16 program [49 ]. The structures of the photoproducts were optimized at the DFT B3LYPD3/6-311++G(2d,2p) level [50 (link),51 (link)], and GD3 dispersion correction was used. The literature reports that, despite its popularity, the global hybrid functional B3LYP has its limitations (errors in the potential energy surface and in the harmonic approximation used) [52 (link)]. However, B3LYP (or rather, its dispersion-corrected versions) is functional, with a proven usefulness for the computation of IR spectra [53 (link),54 (link)]. Taking into account our experience with DFT calculations for small organic compounds and literature studies on the applicability of various functionals, we chose B3LYPD3 to simulate the IR spectra of photoproducts and compare them with experimental spectra. The force-constant matrices were calculated at the same level for the precursor and the photoproducts to evaluate the harmonic frequencies and zero-point vibrational (ZPE) corrections. The structures of the complexes were also optimized at the B3LYPD3 level, and their binding energies (ΔE^CP) were corrected by means of the Boys–Bernardi full-counterpoise procedure [55 (link)]. The selected structural parameters calculated for all the optimized complexes are collected in Tables S11–S14 in the Supplementary Materials. Anharmonic wavenumbers were calculated for the monomeric species at the same level of theory. The vertical excitation energies were calculated using time-dependent density functional theory, TD-DFT [56 (link),57 (link)].

The plant materials comprised the tetraploid R. pseudoacacia, which was two years old. In the spring, bare-root seedlings were planted in uniform-sized pots (diameter × height × bottom diameter = 20 × 28 × 20 cm), and seedlings with uniform growth were chosen for CO₂ treatment after 2 months of growth under normal conditions. The potted seedlings were placed in a light incubator for 3 days for pre-cultivation to acclimate to the new environment. The soil moisture was maintained at 70% throughout, and the culture conditions were as follows: 16 h of light, 8 h of darkness, temperature set at 25 °C, air humidity at 70%, and light intensity at 6000 Lux. The seedlings undergoing the high CO₂ treatment were divided into a control group (natural conditions, a CO₂ concentration of about 0.031%) and treatment groups (a CO₂ concentration of 5%), and the seedlings were subjected to high CO₂ treatment for 9 days before being incubated again under normal CO₂ concentration for 3 days. On day 6 of CO₂ treatment, a respiration accelerator (pyruvic acid, 0.1 mM) and a respiration inhibitor (salicylhydroxamic acid, 1 mM) were sprayed on the leaves of the tetraploid R. pseudoacacia every 3 h. Morphological observations on leaves were made after 0 d, 3 d, 6 d, 9 d, and 12 d. For each treatment, leaves were collected for physiological and proteomic measurements, with three or six biological replicates.
Here, CK-H₂O: untreated control group; CK-PA: pyruvic acid-treated group; CK-SHAM: salicylhydroxamic acid-treated group; T-H₂O: CO₂-treated group; T-PA: CO₂ co-treated with PA; and T-SHAM: CO₂ co-treated with SHAM.