128 ACTA BOT. CROAT. 82 (2), 2023 Acta Bot. Croat. 82 (2), 128–141, 2023 CODEN: ABCRA 25 DOI: 10.37427/botcro-2023-008 ISSN 0365-0588 eISSN 1847-8476 Sugar beet cells’ cellular and extracellular events taking place in response to drought and salinity Dubravko Pavoković1*, Anita Horvatić2, Ingrid Tomljanović3, Biljana Balen1, Marijana Krsnik-Rasol1 1 University of Zagreb, Faculty of Science, Department of Biology, Horvatovac 102a, 10000 Zagreb, Croatia 2 University of Zagreb, Faculty of Food Technology and Biotechnology, Department of Chemistry and Biochemistry, 10000 Zagreb, Croatia 3 GenomeScan B.V., Plesmanlaan 1D, 2333 BZ Leiden, Netherlands Abstract – Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To un- derstand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expres- sion of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liq- uid nutrient medium (PG0). Severe stress altered the intracellular concentrations of most of the measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence cate- gories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcrip- tion factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracel- lular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and affected the expression of cellular and extracellular proteins in sugar beet cells. Keywords: extracellular proteins, mannitol, osmotic stress, proteome, salt stress Introduction Salt stress and drought are major abiotic stressors that significantly affect all aspects of plant physiology, resulting in yield losses of more than 50% and a loss of more than $10.3 billion per year (Ma et al. 2020). Future global scenar- ios, envisioned by the Intergovernmental Panel on Climate Change indicate a decrease in precipitation and an increase in evapotranspiration rates (Pörtner et al. 2022). Initially, both stressors cause water deficit in plants, but under pro- longed salinity, plants respond to hyper-ionic and hyper- osmotic stress in addition to dehydration (Chaves et al. 2009). Plant physiological responses to these stressors aim to minimize water deficit and restore ion homeostasis (Ma et al. 2020). Osmotic adjustments are achieved through the synthesis and accumulation of osmoprotective compounds (Chen and Murata 2002), while disturbances caused by ex- cess sodium ions are remedied by changes in the activity and abundance of sodium/proton exchangers (Deinlein et al. 2014). In addition, both salinity and drought can induce the production of reactive oxygen species (ROS) that can cause lipid, protein, and DNA damage, which, however, can be counteracted by complex nonenzymatic antioxidants (glutathione, ascorbate, carotenoids) and antioxidant en- zymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) (Apel and Hirt 2004). Transcriptomics studies in Arabidopsis revealed that 1008 and 1123 mRNAs are regulated in response to water deficit and salt stress, respectively (Apel and Hirt 2004), im- plying that both stresses involve complex processes. In sug- ar beet, an experiment with salinization and alkalinization identified 4773 and 2251 differentially expressed genes in leaves and roots, respectively (Geng et al. 2020). This and other studies identified ROS-scavenging enzymes, ion transporters and channels, proteins involved in signal transduction, and regulatory proteins, kinases, phosphatas- es, and transcription factors responsible for triggering the * Corresponding author e-mail: dubravko.pavokovic@biol.pmf.unizg.hr mailto:dubravko.pavokovic@biol.pmf.unizg.hr SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 129 stress response (Zhu 2002, Yoshida et al. 2014). However, changes in mRNA levels do not correlate well with protein levels, and many gene products undergo posttranslational modifications that can alter protein activity (Varshavsky 1996, Deyholos 2010). Therefore, protein levels must be de- termined directly rather than extrapolated from transcript abundance. A complementary approach is to use proteomic analyses such as two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) to quantify protein abundance and identify it on a large scale (Hajheidari et al. 2005). To date, several papers have been published analys- ing the response to salt and drought stress using proteomic approaches (Hajheidari et al. 2005, Singh et al. 2022). Al- though there are common proteins that are regulated dur- ing salt and drought stress, each plant species has been shown to respond differently to these stressors. The differ- ences between salt-tolerant (halophytes) and salt-sensitive (glycophytes) plants are particularly pronounced (Askari et al. 2006, Zhang et al. 2012). Most proteomic research has focused on changes in the abundance of cellular proteins, while knowledge of stress-induced expression of extracel- lular proteins is limited. Only recently, it has been shown that the extracellular matrix and its constituent proteins are involved in the response to various stressors in rice, poplar and sweet potato (Zhang et al. 2009, Kim et al. 2013). In this study we aimed to gain insight into the cellular and extracellular processes involved in responses to drought and salinity. To this end, we decided to use cells grown in vitro, since they represent a homogeneous system in which all cells are of similar origin and type, and the conditions of plant tissue culture allow the control of stress homogene- ity and the characterization of cell behavior under stress conditions independently of the regulatory systems acting at the whole plant level (Errabii et al. 2007). On the other hand, plants are composed of a number of cell types that exhibit different cellular characteristics leading to different responses to stimuli. The N, HO, and HNO sugar beet cell lines have proven useful as in vitro models for studying epi- genetic mechanisms, cell differentiation, and metabolism in plants (Le Dily et al. 1990, Causevic et al. 2006). The N line is a normal callus dependent on plant growth regulators, in contrast to the autonomous habituated HNO line and the tumorous T line, which is the result of cell transformation with the Agrobacterium tumefaciens Ti plasmid B6S3 (Pavoković et al. 2012b). In this study, we used the differen- tiated N line, which contains mainly parenchyma cells. It is photosynthetic and grows in response to 2,4-dichlorophen- oxyacetic acid and 6-benzylaminopurine. It exhibits normal nuclear morphology and cell wall cellulose deposition (Pavoković et al. 2012b). To investigate the effects of salt- and mannitol-induced stress at the cellular level, we sought to identify stress-related proteins that are differentially ex- pressed in non-stressed and stressed cells. We also deter- mined possible changes in the concentrations of cellular macro- and microelements. In addition, this study was ex- tended by analyzing the expression of stress-related extra- cellular proteins. We report the disruption of macro- and microelement homeostasis as a consequence of stress and the identification of novel proteins in sugar beet as stress- related proteins. Materials and methods Plant material Sugar beet N cell line (Beta vulgaris L. subsp. vulgaris var. altissima Döll) was grown in vitro in modified Gamborg B5 liquid nutrient medium (PG0) (Negrutiu et al. 1975, Pavoković et al. 2007). The growth chamber was maintained at 22 °C and a 16-h photoperiod (80 µmol photons m–2 s–1). Cells were subcultured every two weeks by transferring 10 mL of the old cells into 40 mL of fresh PG0 medium. The suspensions were shaken on a reciprocal shaker at 125 rpm. Experimental conditions and harvesting Salt stress was generated by growing cells in liquid PG0 medium containing 300 mM NaCl, while physiological drought was provoked by growing cells in the same medium containing 600 mM mannitol. Cells were harvested after 72 h of incubation, washed thoroughly with distilled H2O, dried, and rapidly frozen in liquid nitrogen until use. Macro- and microelements analysis Five samples were used to determine macro- and micro- element concentrations. Samples were analyzed by induc- tively coupled plasma atomic emission spectroscopy (ICP- AES) using the Prodigy High Dispersion ICP instrument (Teledyne Leeman Labs, Hudson, NH). ICP multi-element standard solution IV (Merck, Darmstadt, Germany) was used to control plasma positioning and to prepare standard solutions for calibration. All calibration standards were pre- pared by appropriate dilution of standard stock solutions (1 g L-1) in a concentration range from 0.1 to 5.0 mg L-1. Lyoph- ilized samples were dried at a constant temperature of 70 °C for 1 h and then pulverized in a porcelain mortar. An amount of 0.15 g of each dried sample was weighed with an- alytical accuracy and placed in Teflon vials, except for the solutions of the nutrient medium. 4 mL of concentrated ni- tric acid, HNO3, 1.0 mL of hydrogen peroxide, (w = 30%) and 1.0 mL of ultrapure deionized water (R ≈ 18 MΏ) were add- ed and the vials were left open for 30 min. The vials were sealed and placed in a rack holder of a microwave-assisted high-pressure digestion system (Berghof, Germany). Diges- tion was performed in several steps for 40 minutes. After cooling to room temperature, the solutions were filtered, transferred to 10 mL volumetric flasks, and filled to the mark with ultrapure deionized water. All samples were digested and analyzed as duplicates; blanks were also prepared in the same manner as the samples. To verify the accuracy of the digestion procedure, the same digestion scheme was applied to the certified reference material (SRM 1571 – Orchard leaves). The results are presented as mg macroelement per g of dry weight (DW) or µg microelement per g of DW togeth- er with the standard deviation of measurements. PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 130 ACTA BOT. CROAT. 82 (2), 2023 Analysis of cellular proteins The frozen cells were ground to a fine powder in liquid nitrogen using a pre-cooled mortar and pestle. For 2-DE, the phenol extraction protocol was performed according to a pub- lished procedure (Faurobert et al. 2007). Protein concentra- tion was determined by the modified Bradford method using a UV/Vis spectrophotometer UV-4 (Unicam, UK) and bovine serum albumin (BSA) as a standard (Faurobert et al. 2007). The first dimension, isoelectric focusing (IEF), was per- formed using 18 cm long nonlinear, immobilized pH gradi- ent (IPG) strips, pH 3–10, in the IPGphor system (GE Healthcare, USA), according to Pavoković et al. (2012). The IPG strips were stored at -80 °C until use. IPG strips were thawed and incubated for 15 min in a buffer composed of 0.05 M Tris-HCl pH 8.8, 6 M urea, 2% SDS (w/v) containing 130 mM dithiothreitol (DTT) and then for 15 min in a buf- fer of the same composition, but with 135 mM iodoacet- amide instead of DTT. The second dimension was per- formed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described in Pavoković et al. (2012a), using the PROTEAN II xi system (BioRad, USA). Analysis of extracellular proteins Extracellular proteins were harvested from the liquid medium after cells were removed. To remove debris, the medium was filtered through No 1. Whatman filter papers (Whatman, UK) and again through 0.45 µm Millipore fil- ters (Millipore, USA). Proteins were concentrated on Ami- con ultrafiltration devices (Millipore, USA) with the cut-off of 3 kDa. Protein concentration was determined by the Bradford method using a spectrophotometer and a BSA as standard (Bradford 1976). Proteins were mixed with Laemmli buffer (Laemmli 1970) and loaded onto a large vertical electrophoresis system. Electrophoresis was performed for 30 min at 100 V in stacking gel containing 4% T and 2.67% C and then at 220 V in running gel (12% T, 2.67% C) until the bromophenol blue ran off the gel. Gel staining, image acquisition and analysis Protein spots were visualized using Coomassie Brilliant Blue R-250 (CBB) or by silver staining (Blum et al. 1987). Af- ter protein visualization, gels were scanned with a flatbed scanner (HP, USA) at a resolution of 600 dpi and analyzed with Proteomeweaver 2.2 (Definiens, Germany) using the proposed working pipeline. Protein spots were detected us- ing the following parameters: intensity limit, 10000; contrast limit, 50; and radius limit, 10. Statistical analyzes were per- formed using Proteomeweaver software. At least five gels, each from a biological replicate, were used to create the mas- ter gel for the control and for salt and mannitol treatments. In-gel digestion, peptide sample preparation, and peptide mass spectrometry Spots and bands of differentially expressed proteins were excised from the gels with a scalpel and the gel pieces were washed thoroughly using destaining buffer (30% methanol, 10% glacial acetic acid in H2O) and prepared for matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis as de- scribed in Pavoković et al. (2012b). Mass spectra were ob- tained using a MALDI-TOF/TOF MS (4800 Plus MALDI TOF/TOF analyzer, Applied Biosystems Inc., Foster City, CA, USA) equipped with a 200 Hz, 355 nm Nd:YAG laser, operated in the positive ion reflector mode. For protein identification, we applied the global protein server explorer software (version 3.6, Applied Biosystems, USA) for Mascot (Matrix Science version 2.1, UK) search against the National Center for Biotechnology Information protein database (NCBIprot, http:// www.ncbi.nlm.nih.gov/ protein). Bioinformatics The identified proteins were assigned to functional groups according to the MIPS Functional Catalogue FunCat database (Ruepp et al. 2004) and involved mechanisms reported previously in the literature. The target organelle of proteins was predicted using the TargetP web tool ( Emanuelsson et al. 2007), which predicts whether protein sequences contain a mitochondrial target peptide, a chloro- plast transit peptide or a signal peptide for secretion. At the time of the initial experiment, mass spectrometry was used to identify protein sequences derived from organ- isms other than just sugar beet. To expand the disorder analysis to a set of proteins exclusive to this plant, we ob- tained sequences from the recently published sugar beet ge- nome. We performed a similarity search of the initially identified protein sequences against the sugar beet genome assembly RefBeet-1.2 using the built-in BLAST within the Beta vulgaris Resource (http://bvseq.molgen.mpg.de/blast/) (Dohm et al. 2014). The Universal Protein Resource (UniProt) was used for Gene Ontology analysis (GO, http://www.geneontology.org) of all identified proteins. Statistical analysis For the macro- and microelements analysis the results were analyzed with one-way ANOVA using the STATISTICA 13.0 (Stat Soft Inc., USA) software package. Differences between means were considered statistically significant at P < 0.05 (Tukey post hoc test for unequal sample sizes). Statistical significance for protein expression in 2-D gels was calculated when the protein spot was present on at least three gels. Univariate mean differences between the abundance of protein spots in the control gel and in the gel obtained under salt or mannitol stress were examined using the non-parametric Mann-Whitney-Wilcoxon and Kolmogorov-Smirnov tests. Results Disruption of homeostasis of macro- and microelements Salt- and mannitol-induced stress resulted in disruption of cellular cation homeostasis (Tab. 1 and Tab. 2). Depend- SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 131 ing on the stress and element, different accumulation pat- terns were evident. The concentration of Na+ ions was 7.7-fold higher in salt-treated cells than in the control but 8.3-fold lower with the mannitol treatment (Tab. 1). The concentrations of K+ and Ca2+ ions were decreased in both types of stress compared with the control, but this was par- ticularly pronounced for K+ ions in mannitol treatment (Tab. 1). In contrast, the stress did not affect the concentra- tion of Mg2+ ions. Both types of stress altered the concentra- tions of cellular microelements in different ways: NaCl de- creased the content of Fe2+, Cu2+, and especially Mn2+ ions and increased the concentration of Zn2+ ions, whereas man- nitol decreased the concentration of Fe2+, Mn2+, and Zn2+ ions and increased that of Cu2+ ions (Tab. 2). Salt- and mannitol-responsive cellular proteins We applied a proteomic approach to analyze and com- pare the effect of salt and mannitol on the expression profile of sugar beet proteins. Salt stress resulted in statistically dif- ferent regulation of 43 proteins, of which 22 were up-regu- lated and 21 were down-regulated (Fig. 1). Mannitol-in- duced stress affected the expression of 67 proteins, of which Tab. 1. The concentration of macroelements in control cells of the sugar beet N cell line, in cells treated with 300 mM NaCl or 600 mM mannitol, and in modified Gamborg B5 liquid nutrient medium (PG0) measured by inductively coupled plasma atomic emission spectroscopy. Results are mean values ± standard errors (n = 5). DW - dry weight. Sample mg g–1 DW Na K Ca Mg cell – control 15.00 ± 0.08c 54.58 ± 0.90b 4.465 ± 0.03b 2.568 ± 0.01b cell – 300 mM NaCl 116.8 ± 2.56a 39.7 ± 0.87c 1.941 ± 0.12c 2.536 ± 0.12b cell – 600 mM mannitol 1.738 ± 0.02d 21.65 ± 0.48d 1.599 ± 0.03c 2.528 ± 0.08b PG0 medium 72.47 ± 2.01b 222.0 ± 3.6a 76.79 ± 2.20a 56.37 ± 1.5a Tab. 2. The concentration of selected microelements in control cells of the sugar beet N cell line, in cells treated with 300 mM NaCl or 600 mM mannitol, and in modified Gamborg B5 liquid nutrient medium (PG0) measured by inductively coupled plasma atomic emission spectroscopy. Results are mean values ± standard errors (n = 5). DW - dry weight. Sample μg g–1 DW Fe Cu Mn Zn cell – control 330.9 ± 2.12a 4.382 ± 0.13a 17.53 ± 0.13a 46.15 ± 0.13b cell – 300 mM NaCl 258.3 ± 14.04b 2.928 ± 0.33c 9.382 ± 0.53b 46.98 ± 0.66a cell – 600 mM mannitol 164.8 ± 3.64c 11.86 ± 0.33b 14.93 ± 0.26c 35.25 ± 0.59c PG0 medium 330.9 ± 2.12a 4.382 ± 0.13a 17.53 ± 0.13a 46.15 ± 0.13b Fig. 1. Expression patterns of cellular proteins from a sugar beet N cell line exposed to salt stress (300 mM NaCl) for 72 h obtained by two-dimensional electrophoresis (2-DE). In the first dimension (IEF), 300 µg of proteins were resolved in 18 cm IPG strips with a non- linear pH gradient of 3-10. In the second dimension, proteins were separated on a 12% SDS polyacrylamide gel and then stained with Coomassie Brilliant Blue. Black arrows indicate proteins whose expression was statistically different to that of the control sample (see Materials and methods) and were identified using MALDI-TOF/TOF mass spectrometry. PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 132 ACTA BOT. CROAT. 82 (2), 2023 41 were up-regulated and 26 were down-regulated (Fig. 2). Protein spots were excised and subjected to protein identi- fication by MALDI-TOF/TOF MS. Of the proteins that were differentially expressed under salt stress, 25 were success- fully identified, of which 15 were up-regulated and 10 were down-regulated (On-line Suppl. Tab. 1). Identification of proteins that showed differential expression after mannitol stress was successful for 29 of them, of which 18 were up- regulated and 11 were down-regulated (On-line Suppl. Tab. 2). Some salt- and mannitol-responsive spots were not ex- cised due to their low abundance on the gel. To obtain more information about unresolved proteins or those with low scores, we searched for protein homologs using the BLAST tool (Altschul et al. 1990). Functional clas- sification of proteins was performed using the FunCat da- tabase (http://mips.helmholtz-muenchen.de/funcatDB/) (Ruepp et al. 2004). According to this classification, most of the identified proteins were involved in metabolism, energy and cell rescue, defense, and virulence, accounting for ap- proximately 80% of the proteins (Fig. 3). GO analyses revealed that of the proteins that were up- regulated by salt stress, most were associated with the oxi- dative stress response (4 proteins) and the malate metabol- ic process (2 proteins), whereas other biological processes were represented by only one protein (Fig. 4a). Molecular function analysis revealed that most of the up-regulated proteins were related to hem-, ribosome-, and ATP-binding and to L-malate dehydrogenase activity (Fig. 4b) and exert- ed their function in the cytoplasm and chloroplast (7 and 4 proteins, respectively) (Fig. 4c). As for the down-regulated proteins, most of them are involved in the response to un- folded proteins (Fig. 4a) and have functions in ATP binding (Fig. 4b) in the cytoplasm (Fig. 4c). For most of the proteins that were up-regulated on man- nitol, it was not possible to obtain information on the bio- logical processes in which they are involved, but for those Fig. 2. Expression patterns of cellular proteins from a sugar beet N cell line exposed to mannitol stress (600 mM mannitol) for 72 h obtained by two-dimensional electrophoresis (2-DE) (for details see Fig. 1). Black arrows indicate proteins whose expression was statisti- cally different to that of the control sample (see Materials and methods) and were identified using MALDI-TOF/TOF mass spectrometry. Fig. 3. Functional classification of the identified proteins: salt-induced stress proteins (a), mannitol-induced stress proteins (b). The functional grouping of the different proteins was based on FunCat web tool. The numbers on the pie-chart indicate the number of identified proteins belonging to the respective section. SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 133 for which it was possible, GO analysis revealed that they are involved in malate metabolism and photosynthesis, as well as in the response to fungi and oxidative stress (2 proteins in each category) (Fig. 5a). Analysis of molecular function showed that two proteins had L-malate dehydrogenase ac- tivity, whereas other categories were represented by only one Fig. 5. Gene ontology (GO) analysis of cellular proteins from a sugar beet N cell line exposed to mannitol stress (600 mM mannitol) for 72 h: biological process (a), molecular function (b), and cellular compartment (c). GO analysis was derived through Uniprot hit acces- sions. Differently abundant proteins were identified by MALDI-TOF/TOF MS according to the NCBIprot database. Fig. 4. Gene ontology (GO) analysis of cellular proteins from a sugar beet N cell line exposed to salt stress (300 mM NaCl) for 72 h: biological process (a), molecular function (b), and cellular compartment (c). GO analysis was derived through Uniprot hit accessions. Differently abundant proteins were identified by MALDI-TOF/TOF MS according to the NCBIprot database. PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 134 ACTA BOT. CROAT. 82 (2), 2023 protein (Fig. 5b); the majority of the up-regulated proteins exerted their function in the cytoplasm (Fig. 5c). Of the down-regulated proteins, three are involved in glucose me- tabolism and two in protein folding and response to stress (Fig. 5a); their function is mainly in NAD and ATP binding (2 proteins per category) (Fig. 5b) in the cytoplasm and mi- tochondrial matrix (Fig. 5c), whereas other categories are represented by only one protein. to salt and mannitol stress. We found five protein spots (la- belled E1-E5 in Fig. 6) whose expression changed signifi- cantly after salt and mannitol treatment compared with the control. They were excised from the gel and identified using MALDI-TOF/TOF MS. Two of the five proteins with altered expression (labelled E1 and E4 in Fig. 6) were successfully identified. The other three proteins labelled E2, E3, and E5 are unknown (Fig. 6). Salt stress increased the expression of chitinase precursor (E4) and unknown proteins E2 and E3, whereas it down- regulated the expression of β-D-xylosidase 4 (E1) and un- known protein E5. The expression of β-D-xylosidase was also down-regulated by mannitol (On-line Suppl. Tab. 3). GO analyses revealed that three proteins up-regulated by salt stress are involved in cell tip and root growth and carbohydrate metabolism and have a molecular function in binding copper ions and chitin. The proteins down-regu- lated by NaCl belong to arabinan catabolism, with alpha-L- arabinofuranosidase activity. The single mannitol-respon- sive protein that was down-regulated also belongs to arabinan metabolism, with alpha-L-arabinofuranosidase activity (Fig. 7). Discussion In this study, we investigated the cellular processes in sugar beet cells after a brief but severe exposure to salt and mannitol stress. Although sugar beet is an industrially im- portant crop, there is little proteomic and bioinformatic in- formation on the response of this plant to abiotic stress. By using cell lines instead of whole plants, our intention was to eliminate differences in protein expression between differ- ent tissues and focus on the effects of stress on a small num- ber of cell types. We chose to use high concentrations of salt and mannitol to elicit a stronger cellular response. This ap- proach allowed us to identify a number of previously un- known cellular and extracellular proteins involved in the response to salt and drought stress in sugar beet. A dose- dependent response to salt was also observed in the halo- Fig. 6. Expression patterns of extracellular proteins from a sugar beet N cell line cultured for 72 h in modified Gamborg B5 liquid nutrient medium (PG0) with or without the addition of 300 mM NaCl or 600 mM mannitol. Proteins were resolved on a 12% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue. MW – molecular weight markers, 1 – control, 3 – 300 mM NaCl, 5 – 600 mM mannitol. E1-E5 represent proteins submitted to MALDI-TOF/TOF MS for identification. Fig. 7. Gene ontology (GO) analysis of extracellular proteins from a sugar beet N cell line exposed to salt (300 mM NaCl) and mannitol stress (600 mM mannitol) for 72 h: biological process (a) and molecular function (b). Differently abundant proteins were identified by MALDI-TOF/TOF MS according to the NCBIprot database. GO analysis was derived through Uniprot hit accessions. Salt- and mannitol-responsive extracellular proteins Using experiments in cell culture, we investigated whether extracellular proteins are involved in the response SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 135 phyte Suaeda aegyptica, where the number of regulated pro- teins was positively correlated with salt concentration (Askari et al. 2006). Cation homeostasis Salt and mannitol stress caused profound disruption of cation homeostasis in sugar beet cells. Salt-stressed cells in- creased the concentration of Na+ ions, decreased the con- centration of K+ and Ca2+, whereas the concentration of Mg2+ ions did not change. Accumulation of Na+ ions and loss of K+ ions were observed in sugar beet treated with salt and sorbitol (Wu et al. 2014) and in Mammilaria gracilis callus and tumor exposed to NaCl (Balen et al. 2013). The loss of K+ during salt stress appears to be related to antagonism of Na+ and K+ ions at uptake sites or inhibition of K+ uptake at the plasma membrane (Hu and Schmidhalter 2005). This cellular uptake inhibition could lead to the measured lower Na+ and K+ levels and contribute to osmotic adjustment, in whole sugar beet seedlings during drought, for instance (Wu et al. 2014). Lower Ca2+ levels can be explained by the displacement of extracellularly bound Ca2+ ions by Na+ ions and precipitation of Ca2+ under saline conditions. Such Ca2+ deficiency also allows passive uptake of Na+ ions into cells, resulting in growth arrest and changes in morphology ( Cramer et al. 1988). Loss of Ca2+ has been observed upon salt stress in tomato plants and can be alleviated by small heat shock proteins (HSP) (Fu et al. 2016). Salinity affects micronutrients and nutrient solubility differently depend- ing on the plant species (for a detailed discussion, see Hu and Schmidhalter 2005). It appears that high salinity reduces concentrations of these ions, as has been reported for shoots and roots of marigold (Koksal et al. 2016). Mannitol decreased the concentration of all measured macroelements except Mg2+. A strong decrease in the con- centrations of Na+, K+, and Ca2+ ions was also observed up- on mannitol stress in M. gracilis callus (Balen et al. 2013). In addition, a small reduction in the concentration of K+ and Na+ ions and a large reduction in Ca2+ ions were observed in various organs of sugar beet cv. Janus during long-term drought (Choluj et al. 2008). Ca2+ concentration was re- duced in shoots of two drought-sensitive cultivars during both mild and severe short-term drought (Wu et al. 2014). In addition, mannitol decreased Fe, Mn, and Zn microele- ment content but increased Cu content. The decrease in Fe, Mn, and Zn during drought is thought to be due to their lower solubility (Hu and Schmidhalter 2005). However, the decrease in accumulated ions under mannitol stress may be due to decreased ion uptake rather than the decreased ion mobility that occurs in soil-grown plants during drought (Choluj et al. 2008). Proteome analysis We compared the protein expression profile of control cells with that of cells under salt or mannitol stress. An ap- proximately 1.3-fold difference in abundance compared with control was chosen as the threshold for protein signif- icance, consistent with other similar proteomics studies (Parker et al. 2006, Yan et al. 2006, Jiang et al. 2007). We have identified proteins regulated by salt and drought stress such as H+ATPases (Kirsch et al. 1996), malate dehydroge- nases (Song et al. 2001), heat shock proteins, oxidoreduc- tases, and SOD, but also some new proteins that add to the available information on stress-related proteins in sugar beet. Classification using the FunCat tool showed that most of the identified proteins could be classified into three class- es: (1) cell rescue, defense and virulence, (2) metabolism, and (3) energy. Cell rescue, defense and virulence ROS, such as singlet oxygen (1O2), superoxide anion (O2 •-) and hydrogen peroxide (H2O2), are formed in small amounts under normal growth conditions. Under abiotic stress, the production of ROS increases, which can cause damage to biological molecules (Hajheidari et al. 2005). To counteract ROS, plants employ families of enzymes that are part of their antioxidant system (Deyholos 2010). In the present study, a salt-dependent increase in protein abundance was observed for several APX isoforms and glutathione S-trans- ferase (GST) 6, whereas mannitol increased protein abun- dance of the stromal APX isoform, GST F3 and Mn-SOD II. SODs catalyze the dismutation of O2 •- to H2O2 and O2, and form the first line of defense against the toxic effects in- duced by ROS (Salekdeh et al. 2002). H2O2 is then detoxified to H2O by the APX family, which consists of several iso- forms localized in thylakoid and glyoxisome membranes as well as in the chloroplast stroma and cytosol (Gill and Tuteja 2010). In addition, H2O2 is degraded by peroxiredoxins, and the isoform up-regulated in this study is targeted to chloro- plasts, suggesting that ROS production was increased in this organelle (Dietz 2007). Endogenous and xenobiotic toxic compounds can be detoxified by GSTs, which form a large family consisting of forty-seven members identified in the Arabidopsis genome (Gill and Tuteja 2010). The GSTs up-regulated in this study appear to belong to the plant- specific Type I (phi-class), whose function is to counteract oxidative damage induced by herbicides and abiotic stress (Wagner et al. 2002, Gill and Tuteja 2010). Salt- and mannitol-induced stress regulates another large group of proteins: the family of heat shock proteins (HSPs). In plants, HSPs are classified into five families ac- cording to their molecular weight, amino acid sequence ho- mology, and functions: HSP100, HSP90, HSP70, HSP60, and the small HSP. They can be up-regulated under abiotic stress to support proper folding, unfolding and transloca- tion of proteins to target organelles (Gupta et al. 2010). In this study, the chloroplast and mitochondrial HSP isoforms, together with a chloroplast 60 kDa chaperonin subunit, were significantly down-regulated after salt- and mannitol- induced stress, which may be due to the lower protein synthesis rate as reported in barley roots after salt shock ( Hurkman and Tanaka 1987). Down-regulation of chloro- plast HSP70 and HSP90 isoforms has also been reported PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 136 ACTA BOT. CROAT. 82 (2), 2023 during salt stress in Camellia sinensis (Wang et al. 2015, Chen et al. 2018). Moreover, HSP70 was down-regulated in callus and tumor tissues of M. gracillis treated with either NaCl or mannitol, while HSP60, which is critical for achiev- ing native forms of newly synthesized proteins, was also down-regulated in tumor exposed to NaCl (Rogić et al. 2015). HSP70 can inhibit the transcription of other HSPs by binding to the heat shock transcription factor (HSF) (Kim and Schöffl 2002, Oliver et al. 2011). Reducing HSP70 levels allows for increased production of small HSPs, which play an important role in protection against drought and salt stress (Zou et al. 2012). Glyoxalase I (lactoylglutathione lyase) was down-regu- lated in sugar beet cells under salt stress. It is an enzyme in- volved in the detoxification of the cytotoxic compound methylglyoxal (MG), which is a byproduct of glycolysis. This enzyme was also down-regulated in tobacco cells un- der salt stress (Hoque et al. 2008), suggesting that detoxifi- cation of MG via the glyoxalase system was insufficient in cells under salt stress. Another possible explanation could be that the metabolism of triose phosphates and thus the production of MG was reduced in cells under salt stress. Metabolism At the physiological level, salt stress and drought de- crease stomatal conductance and photosynthesis, and in- crease photorespiration (Atkin and Macherel 2009). During detoxification of photorespiration products, there is an in- creased requirement for the oxidation of NADH and regen- eration of NAD+, which occurs in mitochondria using the mitochondrial NADH dehydrogenase type II or the malate/ oxaloacetate shuttle across the inner mitochondrial mem- brane (Atkin and Macherel 2009). A deficiency of NAD+ in the cell is detrimental because an adequate amount of ATP cannot be provided during stress. Therefore, the increased abundance of cytoplasmic and mitochondrial malate dehy- drogenases detected in our study during salt- and mannitol- induced stress serves to ensure ATP and support normal chloroplast function (Liu et al. 2012). An increase in malate dehydrogenase levels was also observed under 150 mM NaCl stress in Arabidopsis, under treatment with 300 mM NaCl in Eremochloa ophiuroides, and during drought in Elymus elongatum (Jiang et al. 2007, Salekdeh et al. 2007, Liu et al. 2012). We observed up-regulation of one isoform of fructose- biphosphate aldolase and down-regulation of another iso- form, aldolase superfamily protein, after salt stress. In plants, there is a cytosolic and a chloroplast isoform of this enzyme (Konishi et al. 2004). We performed protein local- ization estimation based on the target peptide sequence us- ing the TargetP algorithm (Emanuelsson et al. 2007), and the cytosolic isoform was up-regulated, whereas the chlo- roplast isoform was down-regulated. Up-regulation of the cytoplasmic isoform of the enzyme appears to help plants to cope with anaerobic conditions by promoting the func- tion of the glycolytic pathway for ATP synthesis (Konishi et al. 2004). The down-regulation of the chloroplast isoform is likely a consequence of lower photosynthetic rates and the establishment of tolerance following stress induction ( Yamada et al. 2000). Salt stress resulted in up-regulation of putative quinone reductase, an enzyme belonging to the large family of oxi- doreductases that acts on NADH or NADPH with a qui- none or similar compound as acceptor. It appears to be in- volved in the detoxification of reactive carbonyls in plants (Yamauchi et al. 2010). Quinone reductase was also found to be up-regulated after salt stress in tomato (Zhou et al. 2009). We also observed mannitol-induced down-regula- tion of histidinol dehydrogenase, which is part of amino acid metabolism and catalyzes the final step in the produc- tion of histidine from histidinol. Down-regulation of this enzyme has also been reported in Chlamydomonas reinhardtii exposed to heat shock, suggesting that a reduc- tion in protein biosynthesis occurs during abiotic stress, perhaps as a means to reduce problems with de novo protein folding under adverse conditions (Mühlhaus et al. 2011). Energy Four different proteins belonging to ATPases were dif- ferentially regulated after salt stress: proteins belonging to the endoplasmic reticulum (ER), mitochondria, and V-type ATPases were down-regulated, whereas a subunit of the chloroplast ATPase was up-regulated. Transitional ER ATPase provides energy for vesicle budding, which is re- sponsible for transport between ER and Golgi (Zhang et al. 1994). Down-regulation of this enzyme after salt stress could be a consequence of decreased protein synthesis, which then leads to decreased vesicle production and pro- tein transport. V-type and mitochondrial ATPases are im- portant in salt stress to stimulate the transport of Na+ ions in the vacuole and to restore ion balance, respectively (Lehr et al. 1999, Ndimba et al. 2005). However, the beta-subunit of mitochondrial ATP synthase was down-regulated in rice roots under severe salt stress (500 mM for 2 h), which ulti- mately triggered programmed cell death in salt-sensitive rice (Chen et al. 2009). Salt stress up-regulated two proteins involved in photo- synthesis: a β-subunit of chloroplast ATP-synthase (AtpB) and a 23 kDa polypeptide of the oxygen-evolving complex (OEC) (PsbP) of the photosystem II (PSII), whereas manni- tol up-regulated the 33 kDa OEC protein (PsbO). PsbP and PsbO are extrinsic proteins located on the luminal side of the PSII complex in chloroplasts, where they serve to main- tain oxygen evolution at physiological rates and ensure thy- lakoid stability (De Las Rivas et al. 2007). Their increase under salt stress could help maintain sufficient water-split- ting activity or stabilize PSII under these conditions ( Yamauchi and Sugimoto 2010). Up-regulation of PsbP protein has also been observed in Norway spruce needles under mild drought conditions (Blödner et al. 2007). Mannitol up-regulated ferredoxin-NADP+ reductase (FNR), an enzyme that catalyzes the final step of electron SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 137 transfer in PSI and is responsible for NADPH production. A general increase in FNR transcripts was observed in Ara- bidopsis plants exposed to drought stress (Lehtimäki et al. 2010), and it was concluded that FNR is important in redox regulation and in antioxidant mechanisms in chloroplasts during drought stress. Mannitol also up-regulated mitochondrial NADH-ubi- quinone oxidoreductase, also known as Complex I of the respiratory chain. The complex transfers electrons from NADH to coenzyme Q and translocates protons across the inner mitochondrial membrane, contributing to the build- up of the electrochemical potential needed to produce ATP (Brandt 2006). The up-regulation of the enzyme during drought could help mitochondria to restore ROS imbalance after stress (Nwugo and Huerta 2011, Sharma et al. 2011). Mannitol stress resulted in down-regulation of the gly- colysis enzymes glyceraldehyde-3-phosphate dehydroge- nase (GAPDH) and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM). GAPDH, which cata- lyzes the reversible oxidative phosphorylation of glyceral- dehyde-3-phosphate to 1,3-bisphosphoglycerate, requires NAD(P)H for its activity and is normally inhibited when cells are exposed to oxidative stress (Chernyad’ev and Monakhova 2006, Ralser et al. 2007). IPGAM was found to be down-regulated in Thellungiella halophila upon salt stress, likely due to decreased energy metabolism and sub- sequent formation of ROS (Gao et al. 2008). Moreover, expression of both enzymes was also reduced in M. gracilis tumor exposed to salt and mannitol (Rogić et al. 2015). Taken together, these results suggest that primary meta- bolism in exposed tissues was significantly affected by salt- and mannitol-induced stress and that, in response to salinity and osmotic stress, energy metabolism is decreased to reduce the excessive production of ROS that could trigger oxidative stress (Gao et al. 2008). Other categories of proteins The proteasome is a large protein complex that elimi- nates damaged or misfolded proteins in cells. Here, we found down-regulation of the α-subunit of the proteasome 20S complex in response to salt stress. Considering that stress increases the number of misfolded and damaged pro- teins, one would expect an increase in 20S abundance. How- ever, in wheat roots, 20S proteasome subunit abundance decreased under salt stress, although activity increased (Shi et al. 2011), suggesting that regulation of proteasome activ- ity is not always well correlated with the stress severity. In addition, the 26S proteasome subunit α was found to be down-regulated in M. gracillis tumor tissue treated with NaCl (Rogić et al. 2015), and the authors speculated that under stress, plants process misfolded proteins mainly by refolding. Proteasome abundance may also be down-regu- lated during programmed cell death (Kurepa and Smalle 2008). The down-regulation of receptors for activated C kinase 1 (RACK1) by salt stress appears to be related to its regula- tion by the stress hormone abscisic acid (ABA). RACK1, considered a versatile scaffold protein, is a negative regula- tor of ABA responses. Induction of ABA during salt and drought stress down-regulated the expression of three RackK1 isoforms in Arabidopsis (Guo et al. 2009a). ABA can modulate gene expression depending on whether or not new protein synthesis is required (Zhang et al. 2006). Group A of the bZIP family of transcription factors, the ABRE- BINDING PROTEIN/FACTOR (AREB/ABF) family, is re- sponsible for gene expression that has a ABA-responsive cis- element (ABRE) in its promoter domain (Kim et al. 2011). The bZIP transcription factor up-regulated in this study may be a member of the AREB family in sugar beet respon- sible for mediating ABA responses. Mannitol up-regulated a glycine-rich RNA-binding pro- tein. The superfamily of glycine-rich proteins is involved in post-transcriptional processes such as mRNA and rRNA processing, RNA export, and stability (Hu et al. 2011). A β-subunit of a probable voltage-gated potassium channel localized in the plasma membrane was strongly up-regulat- ed by mannitol. In mammals, β-subunits are not directly involved in potassium transport but in modulating channel activity and are stoichiometrically related to α-subunits transporting ions according to the formula α4β4 (Capera et al. 2019). Ardie et. al. (Ardie et al. 2011) used a β-subunit of a K+ channel from the halophyte Puccinellia tenuiflora and induced it transiently in yeast and Arabidopsis. The activ- ity of the protein can alter the levels of K+ and Na+ in plant parts. During drought, cells suffer from a deficiency of K+ ions (Hu and Schmidhalter 2005) and plants try to maintain a high K+:Na+ ratio to ensure the integrity of cell mem- branes. These results suggest that in sugar beet cells, as in mammals, β-subunits may be involved in modulating K+ channel activity during stress. Extracellular proteins In this study, we observed only five extracellular pro- teins with altered expression. Although the number is lower than reported in other studies (Zhang et al. 2009, Pechanova et al. 2010), this is the first analysis of this type in sugar beet. A β-xylosidase was down-regulated during salt- and man- nitol-induced stress. This enzyme belongs to a family of exo-hydrolases but also has significant transglycosylase activity (Franková and Fry 2011). It is involved in the degradation/reconstruction of xylose-containing poly- saccharides in the plant cell wall during various physio- logical events (Franková and Fry 2011). The down-regula- tion of the enzyme during elevated salinity and drought appears to be part of a defense mechanism in which no cell wall modification serves to alleviate cell damage. AT1G76160 [Arabidopsis thaliana] L-ascorbate oxidase (SKU5 similar protein 5, SKS5), hypothetical protein At1g41830 (SKU5 similar proteins 6, SKS6), and a chitinase were up-regulated only during salt stress. SKS5 and SKS6 belong to a family of multicopper oxidase-like proteins related to ferroxidases, ascorbate oxidases, and laccases PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 138 ACTA BOT. CROAT. 82 (2), 2023 ( Jacobs and Roe 2005). SKS6 is involved in the formation of vascular patterns in cotyledons during Arabidopsis devel- opment, and is positively influenced by plant hormones such as ABA, 1-aminocyclopropane-1-carboxylic acid (ACC, the direct precursor of ethylene), indole-3-acetic acid (IAA, the most common naturally occurring plant hormone of the auxin class), and 2,4-dichlorophenoxyacetic acid ( 2,4-D, a synthetic auxin) (Jacobs and Roe 2005). The role of SKS5 in the cell wall is not known but may be related to hyperhydric stress (Sen and Alikamanoglu 2013). The chitinase protein has a domain of glycoside hydrolase 19 (GH19) and belongs to Class IV chitinase by size. While the primary role of this class of chitinases is in defense against pathogens, up-regulation at the transcriptional level has been observed in Arabidopsis under salt and drought stress (Takenaka et al. 2009, Vaghela et al. 2022). In conclusion, we showed that both stressors altered ion status in cells. Most of the regulated proteins were respon- sible for alleviating the increased ROS production and cel- lular damage and restoring homeostasis. In addition to pre- viously known cellular proteins commonly identified as stress-responsive, we have identified several new proteins that appear to be involved in the stress response. Their role in abiotic stress in sugar beet requires further investigation. By extending our study to the extracellular space, we also identified several extracellular proteins involved in the stress response. Acknowledgments This work was supported by a grant no. 119-1191196- 1200 to prof. dr. sc. Marijana Krsnik-Rasol, a grant “Life under stress: molecular components and mechanisms of plant response to drought and salinity stress” to prof. dr. sc. Marijana Krsnik-Rasol and prof. Dudy Bar-Zvi and a grant “Mechanisms of plant defenses to abiotic stress: changes of proteome due to salt and osmotic stress” to prof. dr. sc. Biljana Balen. Grants are from the Ministry of Science and Education of the Republic of Croatia and University of Zagreb, Croatia. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J., 1990: Basic local alignment search tool. Journal of Molecular Biology 215(3), 403–410. https://doi.org/10.1016/s0022- 2836(05)80360-2 Apel, K., Hirt, H., 2004: Reactive oxygen species: Metabolism, ox- idative stress, and signal transduction. Annual Review of Plant Biology 55, 373–399. https://doi.org/10.1146/annurev.ar- plant.55.031903.141701 Ardie, S. W., Nishiuchi, S., Liu, S., Takano, T., 2011: Ectopic expres- sion of the K+ channel β subunits from puccinellia tenuiflora (KPutB1) and rice (KOB1) alters K + homeostasis of yeast and Arabidopsis. Molecular biotechnology 48(1), 76–86. https://doi. org/10.1007/s12033-010-9349-3 Askari, H., Edqvist, J., Hajheidari, M., Kafi, M., Salekdeh, G. H., 2006: Effects of salinity levels on proteome of Suaeda aegypti- aca leaves. Proteomics 6(8), 2542–2554. https://doi.org/10.1002/ pmic.200500328 Atkin, O.K., Macherel, D., 2009: The crucial role of plant mito- chondria in orchestrating drought tolerance. Annals of Botany 103(4), 581–597. https://doi.org/10.1093/aob/mcn094 Balen, B., Tkalec, M., Rogić, T. , Šimac, M., Peharec Štefanić, P., Rončević, S., Pitarević Svedružić, L., Krsnik-Rasol, M., 2013: Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfei- ff. in vitro-grown cultures. In Vitro Cellular & Developmental Biology – Plant 49, 421–432. https://doi.org/10.1007/s11627- 013-9523-y Blödner, C., Majcherczyk, A., Kües, U., Polle, A., 2007: Early drought-induced changes to the needle proteome of Norway spruce. Tree Physiology 27(10), 1423–1431. https://doi. org/10.1093/treephys/27.10.1423 Blum, H., Beier, H., Gross, H. J., 1987: Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electro- phoresis 8(2): 93–99. https://doi.org/10.1002/elps.1150080203 Bradford, M. M., 1976: A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Analytical Biochemistry 72(1–2), 248–254. https://doi.org/10.1006/abio.1976.9999 Brandt, U., 2006: Energy converting NADH: Quinone oxidoreduc- tase (Complex I). Annual Review of Biochemistry 75, 69–92. https://doi.org/10.1146/annurev.biochem.75.103004.142539 Capera, J., Serrano-Novillo, C., Navarro-Pérez, M., Cassinelli, S., Felipe, A., 2019: The potassium channel odyssey: mechanisms of traffic and membrane arrangement. International Journal of Molecular Sciences 20(3), 734. https://doi.org/10.3390/ ijms20030734 Causevic, A., Gentil, M. V., Delaunay, A., El-Soud, W. A., Garcia, Z., Pannetier, C., Brignolas, F., Hagège, D., Maury, S., 2006: Relationship between DNA methylation and histone acetyla- tion levels, cell redox and cell differentiation states in sugarbeet lines. Planta 224, 812–827. https://doi.org/10.1007/s00425-006- 0267-3 Chaves, M. M., Flexas, J., Pinheiro, C., 2009: Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany 103(4):, 551–560. https://doi. org/10.1093/aob/mcn125 Chen, J., Gao, T., Wan, S., Zhang, Y., Yang, J., Yu, Y., Wang, W., 2018: Genome-wide identification, classification and expres- sion analysis of the HSP gene superfamily in tea plant ( Camellia sinensis). International Journal of Molecular Sciences 19(9), 2633. https://doi.org/doi:10.3390/ijms19092633 Chen, T. H. H., Murata, N., 2002: Enhancement of tolerance of abi- otic stress by metabolic engineering of betaines and other com- patible solutes. Current Opinion in Plant Biology 5(3), 250–257. https://doi.org/10.1016/S1369-5266(02)00255-8 Chen, X., Wang, Y., Li, J., Jiang, A., Cheng, Y., Zhang, W., 2009: Mitochondrial proteome during salt stress-induced pro- grammed cell death in rice. Plant Physiology and Biochemistry 47(5),: 407–415. https://doi.org/10.1016/j.plaphy.2008.12.021 Chernyad’ev, I., Monakhova, O., 2006: Activity of NADP-depen- dent glyceraldehyde-phosphate dehydrogenase and phospho- enolpyruvate carboxylase in wheat leaves under water stress. Applied Biochemistry and Microbiology 42, 312319. https:// doi.org/10.1134/s0003683806030161 Choluj, D., Karwowska, R., Ciszewska, A., Jasinska, M., 2008: In- fluence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants. Acta Physiologiae Plan- tarum 30, 679–687. https://doi.org/10.1007/s11738-008-0166-2 Cramer, G.R., Epstein, E., Läuchli, A., 1988: Kinetics of root elon- gation of maize in response to short-term exposure to NaCl https://doi.org/10.1146/annurev.arplant.55.031903.141701 https://doi.org/10.1146/annurev.arplant.55.031903.141701 https://doi.org/10.1007/s12033-010-9349-3 https://doi.org/10.1007/s12033-010-9349-3 https://doi.org/10.1002/pmic.200500328 https://doi.org/10.1002/pmic.200500328 https://doi.org/10.1093/aob/mcn094 https://doi.org/10.1007/s11627-013-9523-y https://doi.org/10.1007/s11627-013-9523-y https://doi.org/10.1093/treephys/27.10.1423 https://doi.org/10.1093/treephys/27.10.1423 https://doi.org/10.1002/elps.1150080203 https://doi.org/10.1006/abio.1976.9999 https://doi.org/10.1146/annurev.biochem.75.103004.142539 https://doi.org/10.3390/ijms20030734 https://doi.org/10.3390/ijms20030734 https://doi.org/10.1093/aob/mcn125 https://doi.org/10.1093/aob/mcn125 https://doi.org/doi:10.3390/ijms19092633 https://doi.org/10.1016/S1369-5266(02)00255-8 https://doi.org/10.1016/j.plaphy.2008.12.021 https://doi.org/10.1134/s0003683806030161 https://doi.org/10.1134/s0003683806030161 https://doi.org/10.1007/s11738-008-0166-2 SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 139 and elevated calcium concentration. Journal of Experimental Botany 39(11), 15131522. https://doi.org/10.1093/jxb/39.11.1513 De Las Rivas, J., Heredia, P., Roman, A., 2007: Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): Bioinformatic and functional analysis. Biochimica et Biophysica Acta (BBA) – Bioenergetics 1767(6): 575–582. https://doi.org/10.1016/j.bbabio.2007.01.018 Deinlein, U., Stephan, A.B., Horie, T., Luo, W., Xu, G., Schroeder, J. I., 2014: Plant salt-tolerance mechanisms. Trends in Plant Sci- ence 19(6), 371–379. https://doi.org/10.1016/j.tplants.2014.02.001 Deyholos, M. K., 2010: Making the most of drought and salinity transcriptomics. Plant, Cell & Environment 33(4), 648–654. https://doi.org/10.1111/j.1365-3040.2009.02092.x Dietz, K.-J., 2007: The dual function of plant peroxiredoxins in an- tioxidant defence and redox signaling, in: Flohé, L., Harris, J. R. (eds.), Peroxiredoxin systems: Structures and functions, 267–294. Springer, Netherlands. https://doi.org/10.1007/978-1- 4020-6051-9_13 Dohm, J. C., Minoche, A. E., Holtgräwe, D., Capella-Gutiérrez, S., Zakrzewski, F., Tafer, H., Rupp, O., Sörensen, T. R., Stracke, R., Reinhardt, R., Goesmann, A., Kraft, T., Schulz, B., Stadler, P. F., Schmidt, T., Gabaldón, T., Lehrach, H., Weisshaar, B., Him- melbauer, H., 2014: The genome of the recently domesticated crop plant sugar beet (Beta vulgaris). Nature 505, 546–549. https://doi.org/10.1038/nature12817 Emanuelsson, O., Brunak, S., von Heijne, G., Nielsen, H., 2007: Lo- cating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols 2, 953–971. https://doi.org/10.1038/ nprot.2007.131 Errabii, T., Gandonou, C.B., Essalmani, H., Abrini, J., Idaomar, M., Skali Senhaji, N., 2007: Effects of NaCl and mannitol induced stress on sugarcane (Saccharum sp.) callus cultures. Acta Phys- iologiae Plantarum 29, 95–102. https://doi.org/10.1007/s11738- 006-0006-1 Faurobert, M., Pelpoir, E., Chaïb, J., 2007: Phenol extraction of pro- teins for proteomic studies of recalcitrant plant tissues. Meth- ods in Molecular Biology (Clifton, N.J.) 355, 9–14. https://doi. org/10.1385/1-59745-227-0:9 Franková, L., Fry, S. C., 2011: Phylogenetic variation in glycosidas- es and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans-β-xylanase activi- ties. Plant Journal 67(4), 662–681. https://doi.org/10.1111/j. 1365-313X.2011.04625.x Fu, C., Liu, X. X., Yang, W. W., Zhao, C. M., Liu, J., 2016: Enhanced salt tolerance in tomato plants constitutively expressing heat- shock protein in the endoplasmic reticulum. Genetics and Mo- lecular Research 15(2), gmr15028301. https://doi.org/10.4238/ gmr.15028301 Gao F., Zhou Y., Huang L., He D., Zhang G., 2008: Proteomic anal- ysis of long-term salinity stress-responsive proteins in Thellungiella halophila leaves. Chinese Science Bulletin 53(22), 3530–3537. https://doi.org/10.1007/s11434-008-0455-6 Geng, G., Li, R., Stevanato, P., Lv, C., Lu, Z., Yu, L., Wang, Y., 2020: Physiological and transcriptome analysis of sugar beet reveals different mechanisms of response to neutral salt and alkaline salt stresses. Frontiers in Plant Science 11, 571864. https://doi. org/10.3389/fpls.2020.571864 Gill, S. S., Tuteja, N., 2010: Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Phys- iology and Biochemistry 48(12), 909–930. https://doi. org/10.1016/j.plaphy.2010.08.016 Guo, J., Wang, J., Xi, L., Huang, W. D., Liang, J., Chen, J. G., 2009: RACK1 is a negative regulator of ABA responses in Arabidop- sis. Journal of Experimental Botany 60(13), 3819–3833. https:// doi.org/10.1093/jxb/erp221 Gupta, S. C., Sharma, A., Mishra, M., Mishra, R. K., Chowdhuri, D. K., 2010: Heat shock proteins in toxicology: How close and how far? Life Sciences 86(11–12), 377–384. https://doi. org/10.1016/j.lfs.2009.12.015 Hajheidari, M., Abdollahian-Noghabi, M., Askari, H., Heidari, M., Sadeghian, S. Y., Ober, E. S., Salekdeh, G. H., 2005: Proteome analysis of sugar beet leaves under drought stress. Proteomics 5(4), 950–960. https://doi.org/10.1002/pmic.200401101 Hoque, M. A., Banu, M. N. A., Nakamura, Y., Shimoishi, Y., Murata, Y., 2008: Proline and glycinebetaine enhance antioxi- dant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells. Journal of Plant Physiology 165(8), 813–824. https://doi.org/10.1016/j. jplph.2007.07.013 Hu, X., Lu, M., Li, C., Liu, T., Wang, W., Wu, J., Tai, F., Li, X., Zhang, J., 2011: Differential expression of proteins in maize roots in response to abscisic acid and drought. Acta Physiologiae Plan- tarum 33, 24372446. https://doi.org/10.1007/s11738-011-0784-y Hu, Y., Schmidhalter, U., 2005: Drought and salinity: A compari- son of their effects on mineral nutrition of plants. Journal of Plant Nutrition and Soil Science 168(4), 541–549. https://doi. org/10.1002/jpln.200420516 Hurkman, W. J., Tanaka, C. K., 1987: The effects of salt on the pat- tern of protein synthesis in barley roots. Plant Physiology 83, 517–524. https://doi.org/10.1104/pp.83.3.517 Jacobs, J., Roe, J., 2005: SKS6 , a multicopper oxidase-like gene, par- ticipates in cotyledon vascular patterning during Arabidopsis thaliana development. Planta 222, 652–666. https://doi. org/10.1007/s00425-005-0012-3 Jiang, Y., Yang, B., Harris, N. S., Deyholos, M. K., 2007: Compara- tive proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. Journal of Experimental Botany 58(13), 3591–3607. https://doi.org/10.1093/jxb/erm207 Kim, B. H., Schöffl, F., 2002: Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins. Journal of Experimental Botany 53(367), 371–375. https://doi. org/10.1093/jexbot/53.367.371 Kim, J.-S., Mizoi, J., Yoshida, T., Fujita, Y., Nakajima, J., Ohori, T., Todaka, D., Nakashima, K., Hirayama, T., Shinozaki, K., Yamaguchi-Shinozaki, K., 2011: An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulat- ing drought-inducible genes in Arabidopsis. Plant and Cell Physiology 52(12), 2136–2146. https://doi.org/10.1093/pcp/ pcr143 Kim, Y. H., Jeong, J. C., Lee, H. S., Kwak, S. S., 2013: Comparative characterization of sweetpotato antioxidant genes from ex- pressed sequence tags of dehydration-treated fibrous roots un- der different abiotic stress conditions. Molecular Biology Re- ports 40, 2887–2896. https://doi.org/10.1007/s11033- 012-2304-6 Kirsch, M., Zhigang, A., Viereck, R., Low, R., Rausch, T., 1996: Salt stress induces an increased expression of V-type H+-ATPase in mature sugar beet leaves. Plant Molecular Biology 32, 543–547. https://doi.org/10.1007/BF00019107 Koksal N., Alkan-Torun A., Kulahlioglu I., Ertargin E., Karalar E., 2016: Ion uptake of marigold under saline growth conditions. SpringerPlus 5(1), 139. https://doi.org/10.1186/s40064-016- 1815-3 Konishi, H., Yamane, H., Maeshima, M., Komatsu, S., 2004: Char- acterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling. Plant Molecular Biology 56, 839–848. https://doi.org/10.1007/s11103-004-5920-2 Kurepa, J., Smalle, J. A., 2008: Structure, function and regulation of plant proteasomes. Biochimie 90(2), 324–335. https://doi. org/10.1016/j.biochi.2007.07.019 Laemmli, U. K., 1970: Cleavage of structural proteins during as- sembly of head of bacteriophage-T4. Nature 227, 680–685. https://doi.org/10.1038/227680a0 https://doi.org/10.1093/jxb/39.11.1513 https://doi.org/10.1016/j.bbabio.2007.01.018 https://doi.org/10.1016/j.tplants.2014.02.001 https://doi.org/10.1111/j.1365-3040.2009.02092.x https://doi.org/10.1007/978-1-4020-6051-9_13 https://doi.org/10.1007/978-1-4020-6051-9_13 https://doi.org/10.1038/nature12817 https://doi.org/10.1038/nprot.2007.131 https://doi.org/10.1038/nprot.2007.131 https://doi.org/10.1007/s11738-006-0006-1 https://doi.org/10.1007/s11738-006-0006-1 https://doi.org/10.1385/1-59745-227-0:9 https://doi.org/10.1385/1-59745-227-0:9 https://doi.org/10.1111/j.1365-313X.2011.04625.x https://doi.org/10.1111/j.1365-313X.2011.04625.x https://doi.org/10.4238/gmr.15028301 https://doi.org/10.4238/gmr.15028301 https://doi.org/10.3389/fpls.2020.571864 https://doi.org/10.3389/fpls.2020.571864 https://doi.org/10.1016/j.plaphy.2010.08.016 https://doi.org/10.1016/j.plaphy.2010.08.016 https://doi.org/10.1093/jxb/erp221 https://doi.org/10.1093/jxb/erp221 https://doi.org/10.1016/j.lfs.2009.12.015 https://doi.org/10.1016/j.lfs.2009.12.015 https://doi.org/10.1002/pmic.200401101 https://doi.org/10.1016/j.jplph.2007.07.013 https://doi.org/10.1016/j.jplph.2007.07.013 https://doi.org/10.1007/s11738-011-0784-y https://doi.org/10.1002/jpln.200420516 https://doi.org/10.1002/jpln.200420516 https://doi.org/10.1007/s00425-005-0012-3 https://doi.org/10.1007/s00425-005-0012-3 https://doi.org/10.1093/jxb/erm207 https://doi.org/10.1093/jexbot/53.367.371 https://doi.org/10.1093/jexbot/53.367.371 https://doi.org/10.1093/pcp/pcr143 https://doi.org/10.1093/pcp/pcr143 https://doi.org/10.1007/s11033-012-2304-6 https://doi.org/10.1007/BF00019107 https://doi.org/10.1007/s11103-004-5920-2 https://doi.org/10.1016/j.biochi.2007.07.019 https://doi.org/10.1016/j.biochi.2007.07.019 https://doi.org/10.1038/227680a0 PAVOKOVIĆ D., HORVATIĆ A., TOMLJANOVIĆ I., BALEN B., KRSNIK-RASOL M. 140 ACTA BOT. CROAT. 82 (2), 2023 Le Dily, F., Hagege, D., Billard, J. P., Boucaud, J., Gaspar, T., 1990: Effet du Chlorure de Sodium sur la Croissance et le Potentiel Osmotique de Cals Normaux et Habitués de Betterave Sucrière. Biologia Plantarum 32, 256–265. https://doi.org/10.1007/ BF02886945 Lehr, A., Kirsch, M., Viereck, R., Schiemann, J., Rausch, T., 1999: cDNA and genomic cloning of sugar beet V-type H+-ATPase subunit A and c isoforms: evidence for coordinate expression during plant development and coordinate induction in re- sponse to high salinity. Plant Molecular Biology 39, 463–475. https://doi.org/10.1023/A:1006158310891 Lehtimäki, N., Lintala, M., Allahverdiyeva, Y., Aro, E.-M., Mulo, P., 2010: Drought stress-induced upregulation of components involved in ferredoxin-dependent cyclic electron transfer. Jour- nal of Plant Physiology 167(12), 1018–1022. https://doi. org/10.1016/j.jplph.2010.02.006 Liu, Y., Du, H., He, X., Huang, B., Wang, Z., 2012: Identification of differentially expressed salt-responsive proteins in roots of two perennial grass species contrasting in salinity tolerance. Jour- nal of Plant Physiology 169(2), 117–126. https://doi.org/10.1016/j. jplph.2011.08.019 Ma, Y., Dias, M. C., Freitas, H., 2020: Drought and salinity stress responses and microbe-induced tolerance in plants. Frontiers in Plant Science 11, 591911. https://doi.org/10.3389/ fpls.2020.591911 Mühlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M., 2011: Quantitative shotgun proteomics using a uniform 15N-labeled standard to monitor proteome dynamics in time course ex- periments reveals new insights into the heat stress response of Chlamydomonas reinhardtii. Molecular and Cellular Pro- teomics 10(9), M110.004739. https://doi.org/10.1074/mcp. M110.004739 Ndimba, B. K., Chivasa, S., Simon, W. J., Slabas, A. R., 2005: Iden- tification of Arabidopsis salt and osmotic stress responsive pro- teins using two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 5(16), 4185–4196. https://doi. org/10.1002/pmic.200401282 Negrutiu, I., Beeftink, F., Jacobs, M., 1975: Arabidopsis thaliana as a model system in somatic cell genetics I. Cell and tissue cul- ture. Plant Science Letters 5(5), 293–304. https://doi. org/10.1016/0304-4211(75)90057-7 Nwugo, C. C., Huerta, A. J., 2011: The effect of silicon on the leaf proteome of rice (Oryza sativa L.) plants under cadmium- stress. Journal of Proteome Research 10(2), 518–528. https:// doi.org/10.1021/pr100716h Oliver, M. J., Jain, R., Balbuena, T. S., Agrawal, G., Gasulla, F., Thelen, J. J., 2011: Proteome analysis of leaves of the desicca- tion-tolerant grass, Sporobolus stapfianus, in response to dehydration. Phytochemistry 72(10), 1273–1284. https://doi. org/10.1016/j.phytochem.2010.10.020 Parker, R., Flowers, T. J., Moore, A. L., Harpham, N. V. J., 2006: An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina. Journal of Ex- perimental Botany 57(5), 1109–1118. https://doi.org/10.1093/ jxb/erj134 Pavoković, D., Križnik, B., Krsnik-Rasol, M., 2012. Evaluation of protein extraction methods for proteomic analysis of non- model recalcitrant plant tissues, Croatica Chemica Acta, 85(2), 177–183. https://doi.org/10.5562/cca1804 Pavoković, D., Šola, I., Hagège, D., Krsnik-Rasol, M., 2007: Sugar- induced changes in cellular and extracellular protein and gly- coprotein patterns of sugarbeet cell lines. Acta Botanica Cro- atica 66(2), 127–134. https://hrcak.srce.hr/17180 Pechanova, O., Hsu, C. Y., Adams, J. P., Pechan, T., Vandervelde, L., Drnevich, J., Jawdy, S., Adeli, A., Suttle, J.C., Lawrence, A. M., Tschaplinski, T. J., Seguin, A., Yuceer, C., 2010: Apoplast proteome reveals that extracellular matrix contributes to mul- tistress response in poplar. BMC Genomics 11, 674–696. https://doi.org/10.1186/1471-2164-11-674 Pörtner, H.-O., Roberts, D. C., Poloczanska, E. S., Mintenbeck, K., Tignor, M., Alegría, A., Craig, M., Langsdorf, S., Löschke, S., Möller, V., Okem, A., 2022: IPCC, 2022: Summary for Policy- makers, In: Pörtner, H.-O., Roberts, D. C., Tignor, M. M. B., Poloczanska, E., Mintenbeck, K., Alegría, A., Craig, M., Langsdorf, S., Löschke, S., Möller, V., Okem, A., Rama, B. (eds.), Climate change 2022 – impacts, adaptation and vulnerability. Contribution of Working Group II to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change, 3–33. Cambridge University Press, Cambridge, UK. https://doi. org/10.1017/9781009325844.001 Ralser, M., Wamelink, M., Kowald, A., Gerisch, B., Heeren, G., Struys, E., Klipp, E., Jakobs, C., Breitenbach, M., Lehrach, H., Krobitsch, S., 2007: Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. Journal of Biology 6, 10. https://doi.org/10.1186/jbiol61 Rogić T., Horvatić A., Tkalec M., Cindrić M., Balen B., 2015: Pro- teomic analysis of Mammillaria gracilis Pfeiff. in vitro-grown cultures exposed to iso-osmotic NaCl and mannitol. Plant Cell, Tissue and Organ Culture 122(1), 127–146. https://doi. org/10.1007/s11240-015-0756-9 Ruepp, A., Zollner, A., Maier, D., Albermann, K., Hani, J., Mokrejs, M., Tetko, I., Güldener, U., Mannhaupt, G., Münsterkötter, M., Mewes, H. W., 2004: The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Research 32(18), 5539–5545. https:// doi.org/10.1093/nar/gkh894 Salekdeh, G. H., Gazanchian, A., Hajheidari, M., Sima, N. K., 2007: Proteome response of Elymus elongatum to severe water stress and recovery. Journal of Experimental Botany 58(2), 291–300. https://doi.org/10.1093/jxb/erl226 Salekdeh, G. H., Siopongco, J., Wade, L. J., Ghareyazie, B., Bennett, J., 2002: A proteomic approach to analyzing drought- and salt- responsiveness in rice. Field Crops Research 76(2–3), 199–219. https://doi.org/10.1016/S0378-4290(02)00040-0 Sen, A., Alikamanoglu, S., 2013: Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture. In Vitro Cellu- lar & Developmental Biology – Plant 49, 396–404. https://doi. org/10.1007/s11627-013-9511-2 Sharma, L. K., Fang, H., Liu, J., Vartak, R., Deng, J., Bai, Y., 2011: Mitochondrial respiratory complex I dysfunction promotes tu- morigenesis through ROS alteration and AKT activation. Hu- man Molecular Genetics 20(23), 4605–4616. https://doi. org/10.1093/hmg/ddr395 Shi, C., Zhang, Y., Bian, K., Xu, L., 2011: Amount and activity changes of 20S proteasome modified by oxidation in salt-treat- ed wheat root tips. Acta Physiologiae Plantarum 33, 1227–1237. https://doi.org/10.1007/s11738-010-0652-1 Singh, P. K., Indoliya, Y., Agrawal, L., Awasthi, S., Deeba, F., Dwivedi, S., Chakrabarty, D., Shirke, P. A., Pandey, V., Singh, N., Dhankher, O. P., Barik, S. K., Tripathi, R. D., 2022: Genom- ic and proteomic responses to drought stress and biotechno- logical interventions for enhanced drought tolerance in plants. Current Plant Biology 29, 100239. https://doi.org/10.1016/j. cpb.2022.100239 Song, S., Fredlund, K. M., Moøller, I. M., He, Z., 2001: Effects of dehydration stress on germination behavior and mitochon- drial small HSP in imbibed Beta vulgaris L. seeds. Acta Scien- tiarum Naturalium Universitatis Sunyatseni/Zhongshan Dax- ue Xuebao 40, 79–83 (in Chinese with English abstract). Takenaka, Y., Nakano, S., Tamoi, M., Sakuda, S., Fukamizo, T., 2009: Chitinase gene expression in response to environmental https://doi.org/10.1007/BF02886945 https://doi.org/10.1007/BF02886945 https://doi.org/10.1023/A:1006158310891 https://doi.org/10.1016/j.jplph.2010.02.006 https://doi.org/10.1016/j.jplph.2010.02.006 https://doi.org/10.1016/j.jplph.2011.08.019 https://doi.org/10.1016/j.jplph.2011.08.019 https://doi.org/10.3389/fpls.2020.591911 https://doi.org/10.3389/fpls.2020.591911 https://doi.org/10.1074/mcp.M110.004739 https://doi.org/10.1074/mcp.M110.004739 https://doi.org/10.1002/pmic.200401282 https://doi.org/10.1002/pmic.200401282 https://doi.org/10.1016/0304-4211(75)90057-7 https://doi.org/10.1016/0304-4211(75)90057-7 https://doi.org/10.1021/pr100716h https://doi.org/10.1021/pr100716h https://doi.org/10.1016/j.phytochem.2010.10.020 https://doi.org/10.1016/j.phytochem.2010.10.020 https://doi.org/10.1093/jxb/erj134 https://doi.org/10.1093/jxb/erj134 https://doi.org/10.5562/cca1804 https://doi.org/10.1186/1471-2164-11-674 https://doi.org/10.1186/jbiol61 https://doi.org/10.1093/nar/gkh894 https://doi.org/10.1093/nar/gkh894 https://doi.org/10.1093/jxb/erl226 https://doi.org/10.1016/S0378-4290(02)00040-0 https://doi.org/10.1007/s11627-013-9511-2 https://doi.org/10.1007/s11627-013-9511-2 https://doi.org/10.1093/hmg/ddr395 https://doi.org/10.1093/hmg/ddr395 https://doi.org/10.1007/s11738-010-0652-1 https://doi.org/10.1016/j.cpb.2022.100239 https://doi.org/10.1016/j.cpb.2022.100239 SUGAR BEET CELLS IN ABIOTIC STRESS ACTA BOT. CROAT. 82 (2), 2023 141 stresses in Arabidopsis thaliana: Chitinase inhibitor allosami- din enhances stress tolerance. Bioscience, Biotechnology and Biochemistry 73(5), 1066–1071. https://doi.org/10.1271/ bbb.80837 Vaghela, B., Vashi, R., Rajput, K., Joshi, R., 2022: Plant chitinases and their role in plant defense: A comprehensive review. En- zyme and Microbial Technology 159, 110055. https://doi. org/10.1016/j.enzmictec.2022.110055 Varshavsky, A., 1996: The N-end rule: functions, mysteries, uses. Proceedings of the National Academy of Sciences of the Unit- ed States of America 93(22), 12142–12149. https://doi. org/10.1073/pnas.93.22.12142 Wagner, U., Edwards, R., Dixon, D. P., Mauch, F., 2002: Probing the diversity of the Arabidopsis glutathione S – transferase gene family. Plant Molecular Biology 49, 515–532. https://doi. org/10.1023/a:1015557300450 Wang, J., Meng, Y., Li, B., Ma, X., Lai, Y., Si, E., Yang, K., Xu, X., Shang, X., Wang, H., Wang, D., 2015: Physiological and pro- teomic analyses of salt stress response in the halophyte Halogeton glomeratus. Plant, Cell & Environment 38(4), 655–669. https://doi.org/10.1111/pce.12428 Wu, G.-Q., Wang, C.-M., Su, Y.-Y., Zhang, J.-J., Feng, R.-J., Liang, N., 2014: Assessment of drought tolerance in seedlings of sug- ar beet (Beta vulgaris L.) cultivars using inorganic and organic solutes accumulation criteria. Soil Science and Plant Nutrition 60, 565–576. https://doi.org/10.1080/00380768.2014.921579 Yamada, S., Komori, T., Hashimoto, A., Kuwata, S., Imaseki, H., Kubo, T., 2000: Differential expression of plastidic aldolase genes in Nicotiana plants under salt stress. Plant Science 154(1), 61–69. https://doi.org/10.1016/s0168-9452(00)00188-6 Yamauchi, Y., Hasegawa, A., Taninaka, A., Mizutani, M., Sugimoto, Y., 2010: NADPH-dependent reductases involved in the detoxification of reactive carbonyls in plants. Journal of Bio- logical Chemistry 286(9), 6999–7009 https://doi.org/10.1074/ jbc.M110.202226 Yamauchi, Y., Sugimoto, Y., 2010: Effect of protein modification by malondialdehyde on the interaction between the oxygen- evolving complex 33 kDa protein and photosystem II core pro- teins. Planta 231: 1077–1088. https://doi.org/10.1007/s00425- 010-1112-2 Yan, S. P., Zhang, Q. Y., Tang, Z. C., Su, W. A., Sun, W. N., 2006: Comparative proteomic analysis provides new insights into chilling stress responses in rice. Molecular & Cell Proteomics 5(3), 484–496. https://doi.org/10.1074/mcp.M500251-MCP200 Yoshida, T., Mogami, J., Yamaguchi-Shinozaki, K., 2014: ABA-de- pendent and ABA-independent signaling in response to os- motic stress in plants. Current Opinion in Plant Biology 21, 133–139. https://doi.org/10.1016/j.pbi.2014.07.009 Zhang, H., Han, B., Wang, T., Chen, S., Li, H., Zhang, Y., Dai, S., 2012: Mechanisms of plant salt response: insights from pro- teomics. Journal of Proteome Research 11(1), 49–67. https://doi. org/10.1021/pr200861w Zhang, J. H., Jia, W. S., Yang, J. C., Ismail, A. M., 2006: Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Research 97(1), 111–119. https://doi.org/10.1016/j. fcr.2005.08.018 Zhang, L., Ashendel, C. L., Becker, G. W., Morré, D. J., 1994: Isola- tion and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver. The Jour- nal of Cell Biology 127(6), 1871–1883. https://doi.org/10.1083/ jcb.127.6.1871 Zhang, L., Tian, L. H., Zhao, J. F., Song, Y., Zhang, C.J., Guo, Y., 2009: Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimen- sional electrophoresis. Plant Physiology 149(2), 916–928. https://doi.org/10.1104/pp.108.131144 Zhou, S. P., Sauve, R., Fish, T., Thannhauser, T. W., 2009: Salt-in- duced and salt-suppressed proteins in tomato leaves. The Jour- nal of the American Society for Horticultural Science 134(2), 289–294. https://doi.org/10.21273/JASHS.134.2.289 Zhu, J. K., 2002: Salt and drought stress signal transduction in plants. Annual Review of Plant Biology 53, 247–273. https://doi. org/10.1146/annurev.arplant.53.091401.143329 Zou, J., Liu, C., Liu, A., Zou, D., Chen, X., 2012: Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice. Journal of Plant Physiology 169(6), 628–635. https://doi. org/10.1016/j.jplph.2011.12.014 https://doi.org/10.1271/bbb.80837 https://doi.org/10.1271/bbb.80837 https://doi.org/10.1016/j.enzmictec.2022.110055 https://doi.org/10.1016/j.enzmictec.2022.110055 https://doi.org/10.1073/pnas.93.22.12142 https://doi.org/10.1073/pnas.93.22.12142 https://doi.org/10.1023/a:1015557300450 https://doi.org/10.1023/a:1015557300450 https://doi.org/10.1111/pce.12428 https://doi.org/10.1080/00380768.2014.921579 https://doi.org/10.1016/s0168-9452(00)00188-6 https://doi.org/10.1074/jbc.M110.202226 https://doi.org/10.1074/jbc.M110.202226 https://doi.org/10.1007/s00425-010-1112-2 https://doi.org/10.1007/s00425-010-1112-2 https://doi.org/10.1074/mcp.M500251-MCP200 https://doi.org/10.1016/j.pbi.2014.07.009 https://doi.org/10.1021/pr200861w https://doi.org/10.1021/pr200861w https://doi.org/10.1016/j.fcr.2005.08.018 https://doi.org/10.1016/j.fcr.2005.08.018 https://doi.org/10.1083/jcb.127.6.1871 https://doi.org/10.1083/jcb.127.6.1871 https://doi.org/10.1104/pp.108.131144 https://doi.org/10.21273/JASHS.134.2.289 https://doi.org/10.1146/annurev.arplant.53.091401.143329 https://doi.org/10.1146/annurev.arplant.53.091401.143329 https://doi.org/10.1016/j.jplph.2011.12.014 https://doi.org/10.1016/j.jplph.2011.12.014