**1. Introduction**

Plant growth and productivity are compromised by various abiotic stresses, among which are high and low temperature stress. Even short periods of temperature stress may significantly decrease the yield, especially when it occurs during the crucial stages of plant development [1]. As was predicted, heat waves and other extreme temperature events are to become more intense, frequent and long-lasting

because of global climate change [2]. Thus, thermal stresses must be better understood in the context of the response and adaptation of plants, which may enable crops with improved thermotolerance to be obtained and bred [3].

*Brachypodium distachyon* L. Beauv. (Brachypodium), which is a member of the Pooideae subfamily, is a wild annual grass species that is widespread in the regions of the Mediterranean basin, Western Europe, the Middle East, south-west Asia, north-east Africa, North and South America and Australia. It is closely related to many temperate zone key cereals, including wheat, barley, rye, oats and various forage grasses [4]. Due to its numerous favourable biological features such as a relatively small nuclear genome that ranges from 270 Mb to 350 Mb (depending on the methodology that is used), small stature, self-fertility, a life cycle of less than four months and undemanding growth requirements, *B. distachyon* constitutes an excellent model species [5].

Low-temperature stress results in the downregulation of many photosynthesis-related proteins and, at the same time, the upregulation of the proteins that are involved in reactive oxygen species (ROS) scavenging, redox adjustment, cytoskeletal rearrangements, cryoprotection and cell wall remodelling [6]. Similar results are observed in plants that are stressed by a high temperature [7]. Though the cell wall structure is not primarily altered under heat stress, numerous studies have indicated various changes in its architecture. In low temperature stress, changes in the cell wall rigidity may be an important factor in thermotolerance. Changes in the cell wall are more pronounced in roots because they are more sensitive to temperature stresses than the aerial parts of a plant, though the adverse effect of such stress on leaves directly affects plant productivity. Alterations in the cell wall in response to temperature stress concern cellulose and hemicelluloses biosynthesis, pectin modifications by pectin methylesterases, lignin biosynthesis and changes in the abundance of hydroxyproline-rich glycoproteins (HRGP) [8].

HRGP are usually divided into three complex multigene families: (i) arabinogalactan proteins (AGP); (ii) extensins (EXT); and (iii) proline-rich proteins [9]. AGP are further divided into four sub-families according to their polypeptide core: classical AGP, lysine-rich AGP (Lys-rich AGP), arabinogalactan peptides (AG peptides) and fasciclin-like AGP (FLA) [10]. Typically, AGP are strongly O-glycosylated and most of them have glycosylphosphatidylinositol (GPI) anchors that attach the proteins to the plasma membrane, though some of them can be released into the wall matrix *via* GPI cleavage [11]. In connection with their abundance, ubiquitous presence and localisation, AGP play a crucial role in various biological processes such as cell division, cellular communication, programmed cell death, organ abscission, plant-microbe interactions, plant defence and growth as well as in the reproductive processes [12–14]. A decrease in the amount of AGP has also been linked with the loss of embryogenic potential in callus cultures of *B. distachyon* [15]. Despite many studies on the role of AGP in plant development, our understanding of their role in the reaction of the plant to temperature stress is still quite limited. Recent studies have shown that temperature stress strongly affects the distribution and content of AGP in the stigma and ovule of *Solanum lycopersicum* as well as in banana leaves and roots, which may indicate that AGP are differentially regulated in the response to temperature stress and that their expression and distribution is tissue specific [16–18].

Based on a bioinformatic analysis, EXT were divided into seven classes: classical, short, leucine-rich repeat extensins (LRX), proline-rich extensin-like receptor kinases (PERK), formin-homolog EXT (FH EXT), chimeric and long chimeric EXT. EXT are characterised by the presence of serine, which is followed by three to five proline residues. These prolines are hydroxylated and glycosylated [19]. EXT are known to play important roles in the response to wounding and pathogen infections [20]. This family was also indicated as playing an important role in root-microbe interactions [14,21]. A study on a *B. distachyon* callus showed that one of the chimeric EXT could be considered to be a good marker for embryogenic cells [15]. A chimeric leucine-rich repeat/extensin, LRX1, was shown to be required for root hair morphogenesis in *Arabidopsis thaliana* [22]. However, information on the synthesis and location of extensins in response to temperature stress is scarce.

Thus, the aim of this work was to investigate any changes in the distribution of the epitopes of AGP and EXT in *B. distachyon* leaves through an immunostaining analysis. This approach enabled the distribution of these epitopes and the changes in their leaves that had been stressed by a high or low temperature to be determined. We also determined the level of transcript accumulation of selected genes encoding EXT, EXT-like receptor kinases, and FLA in the leaves of *B. distachyon* that had been stressed by a high or low temperature using RT-qPCR.

### **2. Results**

#### *2.1. Distribution of the Epitopes of AGP and EXT in Leaves in Response to Temperature Stress*

The distribution of the epitopes of AGP (JIM8, JIM13, JIM16, LM2 and MAC207), pectin/AGP (LM6) and EXT (JIM11, JIM12 and JIM20) in the leaves of *B. distachyon* under normal (21 ◦C) and thermal stress conditions (4 and 40 ◦C) was determined. Considering the phenotype, we observed no visible changes induced by the thermal stress. The general anatomy of a *B. distachyon* leaf is shown in Figure 1. In order to present the results clearly, only the antibodies for which changes in their localisation or the intensity of the fluorescence signal in different temperature conditions were observed, are presented in the main text. Figures A1–A5 show the localisation of the remaining epitopes, in which no changes were identified in their response to temperature treatment.

**Figure 1.** A cross-section of a *B. distachyon* leaf through the major vascular bundle (nomenclature according to Botha [23]) that had been stained with a fluorescent brightener (FB). Scale bar: 50 μm.

The occurrence of the epitopes of AGP was mostly associated with the vascular bundle. The JIM8 epitope was present in the walls and cellular compartments of the inner bundle sheath cells and phloem (Figure 2D–F). This epitope also occurred in the sclerenchyma fibres that were located next to the vascular bundle (Figure A1A–C) or were developing at the edge of the leaf blade (Figure A1D–F). The presence and spatial distribution of the JIM8 epitope were diverse at different temperatures. The JIM8 epitope was less represented in the leaves that were growing at a low temperature (Figure 2A–C). However, in the leaves that had been subjected to a temperature of 40 ◦C, an increase in the intensity of fluorescence signal was observed in the walls of phloem cells compared to the leaves that were growing at a low temperature (Figure 2G–I). JIM13 was found at the same locations as the JIM8 epitope (Figure A2A–F) and was additionally detected in the intercellular compartments (under intercellular compartments we

define the localisation of epitope within the cytoplasm endomembrane system or organelles that are associated with the biosynthesis and secretion pathway to the wall, however these are not visible on the light microscope level [24]) of the prickles (Figure A2G–I). There were no changes in the distribution of the JIM13 epitope or in the intensity of the fluorescence signal between the analysed temperatures. The JIM16 epitope in the control leaves was present in a low amount in the intercellular compartments of the inner and outer bundle sheath cells and phloem as well as in the xylem parenchyma (Figure 3D–F). This epitope was not detected in the xylem parenchyma in the leaves that were growing at 40 ◦C (Figure 3G–I) and in the leaves from 4 ◦C, the presence of this epitope was not detected (apart from single dots in the vascular bundle cells; Figure 3A–C). Another AGP epitope, LM2, occurred in the cellular compartments of bundle sheath, phloem and xylem parenchyma in the control leaves (Figure 4D–F). At a low temperature (4 ◦C), the occurrence of this epitope was very low (Figure 4A–C), while in the leaves that were growing at a high temperature, it was more abundant (Figure 4G–I) compared to the control plants (Figure 4D–F). Additionally, LM2, was detected in the intercellular compartments and/or walls of the epidermis and bulliform cells in the leaves from plants that had been subjected to a high temperature (Figure 5A–I). A signal in the mesophyll cells was detected only in the leaves that were growing at 40 ◦C (Figure 5G–I). The MAC207 epitope was present in large amounts in the intercellular compartments and/or walls of the phloem cells, mesophyll cells and bulliform cells (Figure A3A–F). At all of the temperatures, its fluorescence had a similar cellular distribution and intensity. The LM6 epitope was detected abundantly in the phloem, xylem parenchyma and, in lower amounts, in the cellular compartments and/or walls of the outer bundle sheath (Figure 6D–F). The fluorescence signal of this epitope in the leaves that were growing at 40 ◦C was more intense compared to the other temperature treatments (Figure 6G–I vs. Figure 6A–F). Outside the vascular bundle, LM6 was present in the cell walls and/or in the intercellular compartments of the mesophyll cells (Figure A4A–C).

**Figure 2.** Immunolocalisation of the JIM8 epitope (**A**–**I**) in cross-sections of the *B. distachyon* leaves, (**A**–**I**): through the major vascular bundle. (**A**–**C**): 4 ◦C; (**D**–**F**): 21 ◦C; (**G**–**I**): 40 ◦C. Abbreviations: FB fluorescent brightener, ib—inner bundle sheath, ob—outer bundle sheath, ph—phloem, ve—vessels, xp—xylem parenchyma. The green colour shows epitope occurrence. Scale bars: 10 μm.

**Figure 3.** Immunolocalisation of the JIM16 epitope (**A**–**I**) in cross-sections of the *B. distachyon* leaves, (A-I): through the major vascular bundle. (**A**–**C**): 4 ◦C; (**D**–**F**): 21 ◦C; (**G**–**I**): 40 ◦C. Abbreviations: FB—fluorescent brightener, ib—inner bundle sheath, ob—outer bundle sheath, ph—phloem, ve—vessels, xp—xylem parenchyma. The green colour shows epitope occurrence. Scale bars: 10 μm.

**Figure 4.** Immunolocalisation of the LM2 epitope (**A**–**I**) in cross-sections of the *B. distachyon* leaves, (**A**–**I**): through the major vascular bundle. (**A**–**C**): 4 ◦C; (**D**–**F**): 21 ◦C; (**G**–**I**): 40 ◦C. Abbreviations: FB—fluorescent brightener, ib—inner bundle sheath, ob—outer bundle sheath, ph—phloem, ve—vessels, xp—xylem parenchyma. The green colour shows epitope occurrence. Scale bars: 10 μm.

**Figure 5.** Immunolocalisation of the LM2 epitope (**A**–**I**) in cross-sections of the *B. distachyon* leaves, (**A**–**I**): through the mesophyll and bulliform cells. (**A**–**C**): 4 ◦C; (**D**–**F**): 21 ◦C; (**G**–**I**): 40 ◦C. Abbreviations: bc—bulliform cells, ep—epidermis, FB—fluorescent brightener, me—mesophyll, pr—prickle. The green colour shows epitope occurrence. Scale bars: 20 μm.

**Figure 6.** Immunolocalisation of the LM6 epitope (**A**–**I**) in cross-sections of the *B. distachyon* leaves, (**A**–**I**): through the major vascular bundle. (**A**–**C**): 4 ◦C; (**D**–**F**): 21 ◦C; (**G**–**I**): 40 ◦C. Abbreviations: FB—fluorescent brightener, ib—inner bundle sheath, ob—outer bundle sheath, ph—phloem, ve—vessels, xp—xylem parenchyma. The green colour shows epitope occurrence. Scale bars: 10 μm.

All three extensin epitopes that are recognised by the JIM11, JIM12 and JIM20 antibodies were mostly associated with the mesophyll cells. The JIM11 epitope was present only in the mesophyll cell walls (Figure A5A–C). In addition to occurring in the mesophyll cell walls (Figure A5D–F), JIM12 was also found in the walls of some of the outer bundle sheath cells and vessels (Figure A5G–I). The occurrence of the JIM20 epitope was similar to JIM12 (Figure A5J–O), but had an additional location in the walls and/or cellular compartments of the phloem (Figure A5M–O). All three extensin epitopes occurred abundantly and there were no differences in their distribution or in the intensity of the fluorescence signal among the temperatures that were analysed.
