*2.2. Analysis of the Level of Transcript Accumulation of the Genes Encoding the FLA, EXT and EXT-Like Receptor Kinases*

In this study, we determined the level of transcript accumulation of five different genes encoding *FLA* (*Bradi4g34420*, *Bradi2g00220*, *Bradi5g18950*, *Bradi3g39740* and *Bradi2g60270*). The transcript accumulation levels of *Bradi4g34420* and *Bradi2g00220* increased in both temperatures, 4 and 40 ◦C, compared to the control conditions (Figure 7A). The increase in expression of the *Bradi4g34420* gene at 40 ◦C (4.7-fold) was higher than at 4 ◦C (1.9-fold) (Figure 7A). In the case of the *Bradi5g18950* gene, the expression at 4 ◦C was approximately the same as in the control, while its expression at 40 ◦C was 6-fold higher (Figure 7A). A similar pattern of expression was found for the *Bradi3g39740* gene, though there was only a slight (1.7-fold) increase in its expression at 4 ◦C (Figure 7B). Interestingly, there was a dramatic increase (28-fold) in the expression of this gene at 40 ◦C. Notably, the expression of the *Bradi2g60270* gene was only detectable in the leaves at 40 ◦C. Generally, temperature stress resulted in a higher level of transcript accumulation of *FLA*, though the increase was more pronounced at 40 ◦C.

**Figure 7.** Relative level of transcript accumulation of the fasciclin-like AGP (*FLA)* genes: (**A**) *Bradi4g34420*, *Bradi2g00220* and *Bradi5g18950* and (**B**) *Bradi3g39740*. The relative expression levels were normalised to an internal control (*Bradi1g32860*, gene encoding ubiquitin) and calibrated to the control. Asterisks indicate significant differences from the control using the Student's t-test (*p* < 0.05; mean ± SD, *n* = 3).

The level of transcript accumulation of nine different genes encoding EXT and EXT-like receptor kinases were also determined. Each gene was assigned to a group based on its structure: *FH EXT* (*Bradi1g22980*, *Bradi3g59780* and *Bradi4g03720*), *chimeric EXT* (*Bradi4g11250* and *Bradi3g12902*) and *PERK EXT* (*Bradi2g00900*, *Bradi2g49240*, *Bradi1g07010* and *Bradi3g31967*). The level of transcript accumulation of the *Bradi1g22980* gene in the treated plants was not statistically different from the control (Figure 8A). The level of transcript accumulation of two other *FH EXT*, *Bradi3g59780* and *Bradi4g03720* was only statistically higher at 40 ◦C (Figure 8A). Conversely, the level of transcript accumulation of the *chimeric*

*EXT*, *Bradi4g11250*, increased significantly at 4 ◦C, though there was no clear difference in its expression for the *Bradi3g12902* gene (Figure 8B). When considering *PERK*, a higher transcript accumulation of the *Bradi2g00900* gene at 4 ◦C and a higher level of transcript accumulation of the *Bradi2g49240* gene at 4 ◦C and 40 ◦C was determined (Figure 8C). Intriguingly, the expression of the other *PERK* gene, *Bradi3g31967*, was only observed in the temperature-stressed samples (Figure 8D). The distribution of all of the epitopes together with changes in the level of transcript accumulation of the analysed genes are summarised in Figure 9.

**Figure 8.** Relative level of transcript accumulation of the extensins (EXT) genes: (**A**) formin-homolog (FH) EXT: *Bradi1g22980*, *Bradi3g59780*, *Bradi4g03720*, (**B**) chimeric EXT: *Bradi4g11250*, *Bradi3g12902*, (**C**) proline-rich extensin-like receptor kinase (PERK): *Bradi2g00900*, *Bradi2g49240*, *Bradi1g07010* and (**D**) *Bradi3g31967*. The relative expression levels were normalised to an internal control (*Bradi1g32860*, gene encoding ubiquitin) and calibrated to the control. Asterisks indicate significant differences from the control using the Student's t-test (*p* < 0.05; mean ± SD, *n* = 3).

**Figure 9.** Consolidated results of the distribution of the epitopes EXT and AGP in the leaves of *B. distachyon* and changes in the level of transcript accumulation of the analysed genes.

#### **3. Discussion**

Although immunohistochemical analyses are widely used to study changes in the chemical components of the cell wall during different developmental processes, in vivo and in vitro information concerning the presence and distribution of the cell wall proteins in leaves that have been subjected to biotic and/or abiotic stresses are scarce and remain largely unexplored [16,18,25]. Previous studies have primarily focused on the differential expression of AGP in response to temperatures stresses in roots and seedlings; however, the involvement of AGP in the response to temperature stress has rarely been studied in leaves [16]. For example, the transient appearance of two AGP proteins in *Triticum aestivum* in response to a low temperature were observed, thus indicating their involvement in the activation of the plant cell defence [16,26]. In transgenic *A. thaliana* plants, non-classical AGP improved the freezing tolerance of seedlings [27]. Temperature stress is one of the factors that limit plant growth and productivity [2,28]. Therefore, data showing changes in the distribution of individual cell wall components, particularly AGP, in connection with temperature stress, are particularly important as the results can be used in genetic engineering for stress tolerance [29].

In the present study, while the distribution of the epitopes of the analysed AGP and EXT was primarily observed in the major vascular bundles (nomenclature according to Botha [23]) and sclerenchyma cells, in the case of the LM2 and MAC207 epitopes, they were present in the mesophyll and epidermal cells, especially in the bulliform cells. Generally, the results for *B. distachyon* presented here are similar to those that have been described for banana leaves in terms of the distribution of epitopes in leaf tissues [18]. In banana, the JIM8 epitope increased in abundance at lower temperatures, thus indicating its role in the tolerance to strong chilling stress [18]. In the pistils of *Solanum lycopersicum* cv Micro-Tom, a high temperature strongly affected the distribution of the JIM8 epitope, which decreased in the stigma and ovule [17]. These varied results with respect to this and other epitopes mean that further intensive studies are necessary. Moreover, such results may indicate that the changes in the chemical composition of the cell walls in response to temperature stress are species-specific. In banana leaves, the presence of the JIM16 epitope was higher in a tolerant cultivar at low temperature stress, thus suggesting that these epitopes may be involved in determining the tolerance of banana to temperature stress [18]. Among the analysed epitopes, the LM2 epitope was detected in most of the leaf tissues and an increase of the LM2 epitope as a response to high temperature stress was observed. A similar distribution was detected in banana leaves, in which this epitope was found in the phloem, bundle sheath, mesophyll and epidermal cells. Low temperature treatment increased the abundance of this epitope in banana [18]. In the leaves of *Tilia x euchlora*, the LM2 epitope was present in the epidermis, hypodermis and parenchyma cells, although as a response to salt stress [25]. Such results may indicate that this wall epitope may be a marker of the plant response to diverse stresses. The abundant presence of the LM6 antibody in *B. distachyon* leaves was found. As LM6 antibody exhibits a high affinity to the (1-5)-α-l-arabinosyl residues, it detects the (1-5)-α-l-arabinan (a pectin rhamnogalacturonan I side chain), however, it can also bind to some AGP (http://www.plantprobes.net/index.php). Thus, the increase in the fluorescence signal in the xylem parenchyma of the temperature-stressed plants may not necessarily indicate changes in the presence of AGP. The function of the arabinan side chains is not well understood and their roles are postulated to be an involvement in the rehydration of the cell wall and flexibility [30–33]. The more abundant presence of the wall components that are recognised by LM6 antibody, especially in the cytoplasmic compartments, that were observed in our study indicate that leaves react to temperature stress by synthesising and depositing (1-5)-α-l-arabinans into the cell walls.

As has been shown in previous studies, analyses of the distribution and changes in the signal intensity of epitopes can be compared with the expression profiles of the genes encoding the proteins that are targeted by these antibodies [15,34]. In this work, we focused on the FLA that have been implicated in modulation of signalling upstream of cell wall polymer biosynthesis, remodelling, as well as in the stress response as one of the AGP sub-families [35–37]. We found an increase in the level of transcript accumulation of the *FLA* in response to temperature stress, which concurs with the immunohistochemistry observations that have been made for the LM2 antibody. It is worth noting that the increase in the level of transcript accumulation of these genes was more pronounced at the high temperature. However, in wheat, four genes encoding *FLA* had a decreased level of transcript accumulation in response to low temperature stress [38]. Similarly, two other *FLA* genes (*OsFLA1* and *OsFLA4*) were downregulated by cold stress in rice [39]. This may reflect intrinsic differences between the analysed species or might be the result of fragmentariness of the conducted experiments, which only focus on a few of the numerous *FLA* genes that are present in a genome. The *Bradi5g18950* gene, which was analysed in our work, exhibited a higher level of transcript accumulation at the high temperature and it was previously shown to be upregulated in a 30-day-old callus that was characterised by an increased embryogenic potential. Conversely, the *Bradi3g39740* gene was linked with a gradual loss of embryogenic potential in *B. distachyon* [15]. Temperature stresses that are induced by low or high temperatures inhibit water uptake, which immediately leads to a slowing down of leaf growth. This observation was correlated with a loss of turgor in leaf cells and an adjustment of the osmoticum, which enables cells to regain turgor [40]. AGP are well known for their water-holding properties [41]. For example, in the resurrection plants, side chains of pectin are highly enriched in arabinose-rich polymers, including AGP. Their presence can prevent water loss during desiccation [8]. In *Co*ff*ea arabica* plants that had been subjected to heat stress, there were extensive changes in the cell wall of the leaves. The plants accumulated a higher content of arabinose and galactose, which may suggest that the response of coffee leaves to heat stress is related to type II arabinogalactans and pectins. Moreover, during heat stress, the palisade parenchyma cells were more separated and thinner relative to the control, which resulted in a decreased thickness of the leaves [42]. It has been hypothesised that the organs that are susceptible to water loss such as leaves increase the thickness of the cell walls, thereby limiting desiccation through the production of specific molecules such as AGP [16]. Our results seem to support this hypothesis.

Similar to the temperature stress, salt stress results in a decrease of available water due to a reduction in osmotic potential of the soil solution, which leads to a water deficit [8]. AGP have also been shown to play an important part in the salt stress response and an upregulation of *AGP* in salt-adapted tobacco BY-2 cell cultures was observed. It has been proposed that AGP act as a possible sodium carrier via vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells [43]. A significant upregulation of the *FLA* genes in the salt stress response was observed in the roots of *Populus trichocarpa* [44]. Moreover, AGP were found to act as pectin plasticisers [8,45]. As was shown for an *FLA sos5* (*salt-overly sensitive*) mutant of *A. thaliana*, it exhibits a root-swelling phenotype under salt stress [46]. Further studies showed that the SOS5 protein mediates adherence via its interaction with the cell wall pectin [47]. *At-FLA4* is one of the *FLA* genes in *A. thaliana* that encodes the predicted lipid-anchored glycoprotein and it was shown to positively regulate cell wall biosynthesis and root growth by modulating abscisic acid signalling. Moreover, an *At-fla4* mutant was found to be sensitive to the salt stress [37]. It has been suggested that *At-fla4* might interact with the pectin network via the covalent or non-covalent interactions of its glycans. *At-fla4* may mechanically link pectin with the *AtFei1* and *AtFei2* receptor kinases and the plasma membrane, thus contributing to some biophysical properties such as swelling and interpolymer connectivity [48]. As was indicated by another inactivated mutant of *A. thaliana*, the *FLA1* gene is involved in the early events of lateral root and shoot development in tissue cultures [49]. Considering the salt stress response, it is possible that the upregulation of *FLA* and the increased signal intensity of some of the epitopes of AGP during temperature stress may link with other cell wall polymers such as pectins and thus may modulate the signalling pathways. As has been shown by a number of studies on various species, the pectin content increases during cold stress. Conversely, it decreases during heat stress [8].

An immunohistochemical analysis of the distribution of the *EXT* using the JIM11, JIM12 and JIM20 antibodies showed the presence of all of these epitopes in the mesophyll and the JIM12 and JIM20 epitopes in the vessels. Although no immunohistochemical studies targeting the EXT using the JIM11, JIM12 and JIM20 antibodies were done in the leaves, the distribution of these epitopes has been widely studied in the callus embryos and roots [20,50–53]. For example, changes in the signal intensity of the JIM11 and JIM12 antibodies were connected with a gradual loss of embryogenic potential in *B. distachyon* callus cultures [15]. Zhang, et al. [54] showed that the EXT that are recognised by the JIM20 antibody were present in the pollen tubes and transmitting tissue of *Nicotiana tabacum* and that the application of hydroxyproline synthesis inhibitor, 3,4-dehydro-L-proline, decreased pollen tube growth. However, studies dedicated to the role of EXT in abiotic stress and especially temperature stress are still scarce. In our study, while we did not observe any changes in the selected epitopes of the EXT in the mesophyll, outer bundle sheath, phloem or vessels at the level of the immunohistochemical analyses, we found an increase of *EXT* and *EXT-like receptor kinase* level of transcript accumulation in the response to temperature stresses, especially during the high temperature stress. This may be partially explained by the fact that we used only three antibodies that bind to the EXT, but it is possible that the application of other anti-EXT antibodies could reveal some changes. Additionally, the effectiveness of immunohistochemical analyses is limited, since it does not provide sufficient resolution and does not focus on individual genes, as is the case of RT-qPCR-based analyses. Changes in the *EXT* gene expression greatly depended on the class of extensins that were being analysed as was shown for *A. thaliana* plants that had been subjected to low temperature stress [55]. Another transcriptomic analysis of the *A. thaliana* response to cold stress showed the downregulation of one of the *PERK* genes [56]. In our experiment, we found that the *PERK extensin*, *Bradi3g31967*, was only expressed in the stressed leaves. The genes that belong to this class have been found in the apical dominance, floral organ defects and root cell elongation [57]. An increased accumulation level of the *PERK4* gene transcript in *A. thaliana* was observed in response to abscisic acid, which is a key regulator of abiotic stress tolerance in plants [58,59]. *PERK1* mRNA from *Brassica napus* was shown to be dramatically and rapidly accumulated in response to wounding and moderately accumulated in response to infection by the fungal pathogen *Sclerotinia sclerotiorum* [60]. Interestingly, the LRX proteins were found to regulate salt tolerance in *A. thaliana*. A triple mutant in the *LRX* genes exhibited a severe salt hypersensitivity and these genes were determined to be an important sensor of the cell wall integrity signals [61]. Moreover, recent studies have hinted at the role of EXT in the plant defence against phytopathogens as well as in interactions with beneficial microorganisms [14,21,62]. EXT have also been implicated in aluminium resistance and its accumulation in the cell walls of pea roots was observed [63]. The changes in the plant cell wall in response to temperature stress are diverse and not only include AGP and EXT, but also alterations in cellulose, hemicellulose, pectin and lignin biosynthesis [8]. Further investigations into the changes in cell wall proteomes could unravel the involvement of other proteins in the stress response because the proteome of the *B. distachyon* cell walls is complex and consists of at least 594 proteins [64–68].

#### **4. Materials and Methods**

### *4.1. Plant Material*

The plants of the *B. distachyon* reference genotype Bd21 that were used is this experiment were cultivated in pots that had been filled with soil mixed with vermiculite at a ratio of 3:1 in a greenhouse. The seeds of *B. distachyon* genotype Bd21 (accession number: PI 254867) were sourced from the collection held by the United States Department of Agriculture—National Plant Germplasm System. The plants were grown in the greenhouse under a 16 h/8 h light/dark photoperiod at 21 ± 1 ◦C and were illuminated by lamps emitting white light at an intensity of 10 000 lx. For the low temperature stress, the plants were incubated at 4 ◦C for 24 h and for the high temperature stress, the plants were incubated at 40 ◦C for 24 h in growing chambers [69]. Plants at the fourth stage of principal growth according to the Hong, et al. [70] were used in this experiment. This stage is referred to as booting and is characterised by the emergence of the head at the top of the growing shoot. The flag leaf was harvested and used to isolate the RNA and to perform the RT-qPCR analysis. For the immunohistochemistry

analysis, the middle part of the leaf was collected because this permitted clear observations of the major vascular bundle, epidermis, bulliform cells, mesophyll and sclerenchyma.

#### *4.2. Sample Preparation*

To determine the chemistry of the cell wall, a set of monoclonal antibodies against the specific cell wall epitopes of the AGP (antibodies JIM8, JIM13, JIM16, LM2, MAC207), pectin/AGP (LM6) and EXT (JIM11, JIM12 and JIM20) (Plant Probes, Leeds, UK) were used. The references and information on the antibodies are shown in Table 1. The leaves were excised, fixed and embedded in Steedman's wax [20,71]. Transverse sections of the leaf blade (7 μm thick) were cut using a HYRAX M40 rotary microtome (Zeiss, Oberkochen, Germany) and collected on microscopic slides coated with poly-L-lysine (Menzel Gläser, Braunscheig, Germany).

#### *4.3. Immunohistochemistry*

The sections were de-waxed and rehydrated in an ethanol series (three times in 100, 90 and 50% ethanol in phosphate buffered saline PBS, *v*/*v*, each for 10 min) and PBS (10 min) [71]. The detailed procedure for immunochemical analysis and histological section observation was as previously described [20]. The slides were stained with 0.01% (*w*/*v*) fluorescent brightener 28 (FB) (Sigma-Aldrich, St. Louis, MO, USA) in PBS, which was used to visualise cell walls due to its affinity to cellulose. Two biological replicates were performed with at least eight sections for each replicate.

#### *4.4. RT-qPCR*

In order to characterise the level of transcript accumulation of the selected genes, RT-qPCR was performed using a LightCycler® 480 SYBR Green I Master in a LightCycler® 480 Real-Time PCR System (Roche, Basel, Switzerland). The total RNA was isolated from the leaves of *B. distachyon*. The primers used in this research are shown in Table A1. The genes encoding extensins with their division into classes were as previously described [19]. The *FLA* genes were selected based on the annotation found in the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). The detailed procedure for RT-qPCR was as in Betekhtin, et al. [72]. Briefly, the isolated RNA were treated with the DNase (QIAGEN, Hilden, Germany), and subsequently used for first-strand cDNA generation. Samples were run in the LightCycler® 480 Real-Time PCR System (Roche, Basel, Switzerland). The PCR conditions were as follow: 5 min at 95 ◦C, 45 cycles of 10 s at 95 ◦C, 20 s at 60 ◦C and 10 s at 72 ◦C with signal acquisition. Ubiquitin was used as the reference gene and analysis was performed using the 2−ΔΔ*C*<sup>T</sup> method. The significant differences between the samples and control were calculated using the Student's *t*-test.


**Table 1.** The antibodies that were used for the immunocytochemistry, the epitopes they recognise and the relevant references.
