<table><caption>Table 1. Cont.</caption><thead><tr><th>Species</th><th>Acc. No. 1</th><th>Protein 2</th><th>MW 3</th><th>pl 3</th><th>Signal 4</th><th>N-Glyco 5</th><th>NetPhos 6</th><th>PCA 7</th><th>PSORT 8</th><th>Response to</th></tr></thead><tbody><tr><td rowspan="11">O. sativa</td><td rowspan="11">Q5VR15</td><td>MbPrx[J]113</td><td>12.74</td><td>5.56</td><td>1-24</td><td>—</td><td>10</td><td>—</td><td>ER</td><td>cadmium treatment, nodulation, nematode<br>intection and pathogen *</td></tr><tr><td>A2Q887</td><td>MbPrx[J]116</td><td>33.27</td><td>7.98</td><td>1-14</td><td>1</td><td>23</td><td>—</td><td>PM</td><td>nitrogen and phosphate starvation and pathogen *</td></tr><tr><td>G7ICN9</td><td>MbPrx[J]117</td><td>17.60</td><td>6.23</td><td>1-19</td><td>—</td><td>13</td><td>—</td><td>PM</td><td>nitrogen and phosphate starvation and pathogen *</td></tr><tr><td>Q5U175</td><td>O8Prx01</td><td>34.78</td><td>6.88</td><td>1-24</td><td>1</td><td>30</td><td>—</td><td>ER</td><td>—</td></tr><tr><td>Q5U116</td><td>O8Prx07</td><td>31.93</td><td>5.32</td><td>1-20</td><td>1</td><td>27</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q5U114</td><td>O8Prx09</td><td>35.50</td><td>5.67</td><td>1-11</td><td>5</td><td>31</td><td>—</td><td>PM</td><td>etiolation and drought *</td></tr><tr><td>Q5U113</td><td>O8Prx10</td><td>36.20</td><td>5.38</td><td>—</td><td>5</td><td>34</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q5U110</td><td>O8Prx13</td><td>36.18</td><td>5.16</td><td>1-21</td><td>8</td><td>18</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q8ER51</td><td>O8Prx30</td><td>34.43</td><td>7.52</td><td>1-26</td><td>1</td><td>33</td><td>+</td><td>PM</td><td>—</td></tr><tr><td>Q5UIR1</td><td>O8Prx32</td><td>34.06</td><td>8.42</td><td>1-30</td><td>2</td><td>30</td><td>—</td><td>PM</td><td>pathogen *</td></tr><tr><td>Q5UIR7</td><td>O8Prx36</td><td>50.76</td><td>4.85</td><td>1-17</td><td>2</td><td>45</td><td>+</td><td>PM</td><td>nitrogen and phosphate starvation *</td></tr><tr><td>Q5UI17</td><td>O8Prx46</td><td>35.95</td><td>7.02</td><td>1-29</td><td>1</td><td>20</td><td>—</td><td> Vac</td><td>gibberellic acid and heat *</td></tr><tr><td>Q7XU11</td><td>O8Prx556</td><td>51.48</td><td>9.15</td><td>1-26</td><td>5</td><td>86</td><td>—</td><td>PM</td><td>drought *</td></tr><tr><td>Q7XVZ8</td><td>O8Prx65</td><td>37.61</td><td>7.20</td><td>1-24</td><td>2</td><td>21</td><td>—</td><td>MaM</td><td>—</td></tr><tr><td>Q7LW2</td><td>O8Prx63</td><td>37.58</td><td>5.63</td><td>1-28</td><td>6</td><td>38</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q6Z211</td><td>O8Prx10</td><td>32.78</td><td>5.68</td><td>1-20</td><td>2</td><td>27</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q6Z221</td><td>O8Prx111</td><td>32.78</td><td>5.68</td><td>1-20</td><td>2</td><td>27</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>Q8CVG6</td><td>O8Prx106-1</td><td>40.15</td><td>7.22</td><td>1-19</td><td>2</td><td>47</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>A2YP47</td><td>O8Prx106-2</td><td>40.15</td><td>7.22</td><td>1-19</td><td>2</td><td>45</td><td>—</td><td> Vac</td><td>nitrogen and phosphate starvation *</td></tr><tr><td>Q8CVG0</td><td>O8Prx107</td><td>34.23</td><td>9.19</td><td>1-22</td><td>3</td><td>15</td><td>—</td><td>ER</td><td>anaerobic stress, brassinolide and gibberellic acid treatments and<br> nematode infection *</td></tr><tr><td>Q5UI14</td><td>O8Prx109</td><td>33.02</td><td>8.26</td><td>1-21</td><td>4</td><td>32</td><td>+</td><td>ER</td><td>nitrogen and phosphate starvation *</td></tr><tr><td rowspan="11">O. sativa</td><td rowspan="11">Q6Z3Y8</td><td>O8Prx16</td><td>36.05</td><td>7.69</td><td>1-19</td><td>1</td><td>29</td><td>+</td><td>MaM</td><td>pH and oxidative stress *</td></tr><tr><td>Q5UI16</td><td>O8Prx17</td><td>33.51</td><td>6.65</td><td>1-25</td><td>1</td><td>30</td><td>—</td><td>PM</td><td>pH, oxidative stress, nitrogen starvation and pathogen *</td></tr><tr><td>Q6U125</td><td>O8Prx18</td><td>36.93</td><td>4.96</td><td>1-25</td><td>2</td><td>23</td><td>—</td><td> Vac</td><td>—</td></tr><tr><td>Q7X1B1</td><td>O8Prx126</td><td>35.48</td><td>4.45</td><td>1-27</td><td>1</td><td>33</td><td>—</td><td>PM</td><td>pH, oxidative stress, nematode infection and pathogen *</td></tr><tr><td>Q5UI18</td><td>O8Prx135</td><td>34.83</td><td>8.79</td><td>1-31</td><td>3</td><td>38</td><td>+</td><td>PM</td><td>nitrogen starvation *</td></tr><tr><td>Q5UI15</td><td>O8Prx138</td><td>35.66</td><td>8.40</td><td>—</td><td>2</td><td>17</td><td>—</td><td>PM</td><td>nitrogen starvation and pathogen *</td></tr><tr><td rowspan="11">Z. mays</td><td rowspan="11">A0A1D6KU1F3</td><td>ZmPrx01</td><td>38.36</td><td>6.81</td><td>—</td><td>5</td><td>35</td><td>—</td><td>PM</td><td>cell wall modification, wounding and pathogen *</td></tr><tr><td>ZmPrx11</td><td>35.44</td><td>5.14</td><td>—</td><td>3</td><td>28</td><td>+</td><td>PM</td><td>pH, oxidative stress, abscissic and ethylene stress, etiolation and heat<br>shock *</td></tr><tr><td>B4FHG3</td><td>ZmPrx24</td><td>37.82</td><td>5.9</td><td>1-25</td><td>2</td><td>36</td><td>—</td><td>PM</td><td>pH, drought and pathogen *</td></tr><tr><td>B4F228</td><td>ZmPrx26</td><td>37.50</td><td>5.01</td><td>1-38</td><td>3</td><td>34</td><td>—</td><td> MiM</td><td>heat and oxidative stress *</td></tr><tr><td>B6I173</td><td>ZmPrx35</td><td>36.80</td><td>6.05</td><td>1-22</td><td>2</td><td>24</td><td>—</td><td>ER</td><td>heat and oxidative stress *</td></tr><tr><td>A0A1D6H455</td><td>ZmPrx36</td><td>33.09</td><td>4.90</td><td>1-24</td><td>2</td><td>29</td><td>+</td><td>MaM</td><td>pathogen</td></tr><tr><td>B4FVT1</td><td>ZmPrx39</td><td>37.91</td><td>8.30</td><td>1-21</td><td>8</td><td>34</td><td>—</td><td>MaM</td><td>pH, drought and etiolation *</td></tr><tr><td>A0A1D6F816</td><td>ZmPrx41</td><td>35.75</td><td>4.88</td><td>—</td><td>1</td><td>30</td><td>—</td><td>PM</td><td>pathogen *</td></tr><tr><td>Q7ZT56</td><td>ZmPrx46</td><td>38.65</td><td>6.19</td><td>1-23</td><td>1</td><td>33</td><td>—</td><td>ER</td><td>—</td></tr><tr><td>K7YQ80</td><td>ZmPrx52</td><td>34.59</td><td>8.96</td><td>1-27</td><td>4</td><td>29</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>B4L139</td><td>ZmPrx53</td><td>46.55</td><td>4.98</td><td>1-24</td><td>1</td><td>42</td><td>+</td><td> Vac</td><td>cold stress</td></tr><tr><td>A0A1D6M201</td><td>ZmPrx55</td><td>34.96</td><td>4.85</td><td>1-42</td><td>1</td><td>33</td><td>+</td><td>PM</td><td>pathogen *</td></tr><tr><td>B6T589</td><td>ZmPrx57</td><td>34.57</td><td>4.38</td><td>1-52</td><td>1</td><td>36</td><td>—</td><td>PM</td><td>pathogen *</td></tr><tr><td>B4FH68</td><td>ZmPrx58</td><td>36.91</td><td>6.35</td><td>1-25</td><td>1</td><td>30</td><td>—</td><td>PM</td><td>pH, oxidative stress, abscissic and ethylene stress, etiolation and heat<br>shock *</td></tr><tr><td>A5H454-1</td><td>ZmPrx66</td><td>33.42</td><td>8.39</td><td>1-29</td><td>4</td><td>70</td><td>+</td><td>PM</td><td>drought</td></tr><tr><td>A0A1D6H455</td><td>ZmPrx67</td><td>32.77</td><td>4.89</td><td>—</td><td>4</td><td>26</td><td>—</td><td>MaM</td><td>heat, drought, pathogen and oxidative stress *</td></tr><tr><td>A5H452</td><td>ZmPrx70</td><td>33.41</td><td>9.08</td><td>1-25</td><td>4</td><td>34</td><td>+</td><td>ER/PM</td><td>drought</td></tr><tr><td>B4FM18</td><td>ZmPrx71</td><td>35.71</td><td>9.64</td><td>1-28</td><td>3</td><td>30</td><td>—</td><td>PM</td><td>nitrogen starvation *</td></tr><tr><td>B4F779</td><td>ZmPrx72</td><td>36.67</td><td>9.31</td><td>—</td><td>2</td><td>22</td><td>—</td><td> MiM</td><td>drought</td></tr><tr><td>B6SN19</td><td>ZmPrx76</td><td>33.30</td><td>8.64</td><td>1-24</td><td>—</td><td>33</td><td>—</td><td>ER</td><td>nitrogen starvation *</td></tr><tr><td>B4FH58</td><td>ZmPrx77</td><td>35.71</td><td>9.64</td><td>1-32</td><td>6</td><td>20</td><td>+</td><td>ER</td><td>—</td></tr><tr><td>A0A1D6K4W2</td><td>ZmPrx78</td><td>41.42</td><td>5.86</td><td>1-21</td><td>3</td><td>40</td><td>+</td><td> Vac</td><td>pH and drought *</td></tr><tr><td>B4C39</td><td>ZmPrx81</td><td>36.62</td><td>7.66</td><td>1-29</td><td>3</td><td>35</td><td>—</td><td>PM</td><td>drought</td></tr><tr><td>A0A1D6E530</td><td>ZmPrx85</td><td>35.48</td><td>5.35</td><td>1-23</td><td>1</td><td>32</td><td>—</td><td>PM</td><td>pathogen</td></tr></tbody></table>
<table><caption>Table 1. Cont.</caption><thead><tr><th>Species</th><th>Acc. No. 1</th><th>Protein 2</th><th>MW 3</th><th>pl 3</th><th>SignalP 4</th><th>N-Glyco 5</th><th>NetPhos 6</th><th>PCA 7</th><th>PSORT 8</th><th>Response to</th></tr></thead><tbody><tr><td rowspan="10">Z. mapes</td><td>Q9ZT58</td><td>ZmPrs86</td><td>35.51</td><td>4.37</td><td>1-21</td><td>3</td><td>34</td><td>+</td><td>PM</td><td>pathogen</td></tr><tr><td>A0A1D6hQQ9</td><td>ZmPrs89</td><td>36.08</td><td>4.87</td><td>1-32</td><td>10</td><td>23</td><td>+</td><td>MIM</td><td>pH, anoxia, ethylene and gibberellin acid *</td></tr><tr><td>A0A1D6hQQ8</td><td>ZmPrs94</td><td>37.24</td><td>5.82</td><td>1-29</td><td>6</td><td>30</td><td>—</td><td>PM</td><td>drought</td></tr><tr><td>B6TWB1</td><td>ZmPrs102</td><td>37.11</td><td>6.00</td><td>1-26</td><td>4</td><td>24</td><td>—</td><td>Vac</td><td>pH and pathogen *</td></tr><tr><td>A0A1D6IKX3</td><td>ZmPrs103</td><td>57.26</td><td>5.06</td><td>1-27</td><td>4</td><td>106</td><td>—</td><td>Vac</td><td>—</td></tr><tr><td>B4EBH0</td><td>ZmPrs104</td><td>38.04</td><td>8.09</td><td>1-27</td><td>2</td><td>23</td><td>—</td><td>Vac</td><td>etiolation *</td></tr><tr><td>B4FYH1</td><td>ZmPrs109</td><td>37.94</td><td>6.00</td><td>1-22</td><td>3</td><td>40</td><td>—</td><td>PM</td><td>pathogen *</td></tr><tr><td>A0A1D6fJ12</td><td>ZmPrs112</td><td>41.37</td><td>7.03</td><td>1-27</td><td>6</td><td>64</td><td>—</td><td>PM</td><td>etiolation, drought and pathogen *</td></tr><tr><td>A0A1D6KQ20</td><td>ZmPrs113</td><td>38.28</td><td>10.26</td><td>1-23</td><td>3</td><td>27</td><td>—</td><td>PM</td><td>pH, drought and pathogen *</td></tr><tr><td>C0P786</td><td>ZmPrs114</td><td>32.71</td><td>5.20</td><td>1-23</td><td>5</td><td>38</td><td>+</td><td>PM</td><td>etiolation, cell wall modification, wounding and pathogen *</td></tr><tr><td>B616W01</td><td>ZmPrs115</td><td>37.59</td><td>8.31</td><td>7</td><td>3</td><td>40</td><td>+</td><td>PM</td><td>drought *</td></tr><tr><td>A0A1D6H652</td><td>ZmPrs122</td><td>34.37</td><td>6.88</td><td>1-28</td><td>2</td><td>29</td><td>+</td><td>PM</td><td>etiolation, abscisic acid and pathogen *</td></tr><tr><td>B4FA32</td><td>ZmPrs123</td><td>34.01</td><td>6.50</td><td>1-25</td><td>1</td><td>25</td><td>+</td><td>PM</td><td>pH and sulfur deficiency *</td></tr><tr><td>A0A1D6N9NS</td><td>ZmPrs124</td><td>37.42</td><td>4.84</td><td>1-23</td><td>2</td><td>25</td><td>—</td><td>PM</td><td>etiolation, abscisic acid and pathogen *</td></tr><tr><td>K7TMB0</td><td>ZmPrs128</td><td>35.80</td><td>8.05</td><td>1-36</td><td>3</td><td>30</td><td>—</td><td>MoM</td><td>—</td></tr><tr><td>K7U151</td><td>ZmPrs129</td><td>33.75</td><td>6.80</td><td>1-26</td><td>1</td><td>20</td><td>—</td><td>PM</td><td>—</td></tr><tr><td>A0A1D6JNY2</td><td>ZmPrs132</td><td>46.64</td><td>4.99</td><td>1-22</td><td>1</td><td>40</td><td>+</td><td>Vac</td><td>—</td></tr><tr><td>K7VFH6</td><td>ZmPrs135</td><td>35.67</td><td>4.98</td><td>1-31</td><td>—</td><td>22</td><td>—</td><td>ER</td><td>—</td></tr><tr><td>C4IZQ0</td><td>ZmPrs136</td><td>34.48</td><td>8.45</td><td>1-22</td><td>2</td><td>21</td><td>—</td><td>PM</td><td>etiolation, drought and pathogen *</td></tr><tr><td>A0A1D6jF04</td><td>ZmPrs140</td><td>37.88</td><td>8.88</td><td>1-21</td><td>3</td><td>31</td><td>—</td><td>PM</td><td>pH *</td></tr></tbody></table>

<footnote n="1">¹ http://www.uniprot.org/; ² http://peroxibase.toulouse.inra.fr/; ³ https://web.expasy.org/protparam/; ⁴ http://www.cbs.dtu.dk/services/SignalP/; ⁵ http://www.cbs.dtu.dk/services/NetGlyc/; ⁶ http://www.cbs.dtu.dk/services/NetPhos/; ⁷ https://prosite.expasy.org/; ⁸ http://psort1.hgc.jp/form.html.</footnote>
In silico analyzes of putative membrane-bound peroxidases revealed at least one glycosylation-site with the exception of some endoplasmatic reticulum (ER) and vacuolar peroxidases (Table 1). These membrane-bound peroxidases were predicted to be highly phosphorylated. Glycosylation of class III peroxidases seem to be necessary for protein folding, stability and catalytic activity [48]. Glycosylation is necessary for activity of PM-bound peroxidases [22]. Phosphorylation may change properties of the enzymes and be involved in protein-protein interaction. These predictions have to be verified by experimental data.

<figure><img src="image_103.png" /><figcaption>**Figure 2.** Putative tertiary structures of PM-bound class III peroxidases. Structures were predicted by SWISS-MODEL for (a) ZmPrx01 (3hdlA, 1 TMH, root PM), (b) OsPrx95 (3hdl1A, 3 TMH, root PM), (c) AtPrx64 (3hdl1A, 0 TMH, PM), (d) AtPrx47 (5twt, 1 TMH, PM), and (e) MtPrx02 (5twt1A, 1 TMH, root tip DRM). N-terminal amino acid sequences containing transmembrane domains (marine) were modeled by ab initio with Phyre2 in intensive mode [49]. Ligand binding-sites were predicted by 3DLigandSite [50]. The hypothetical models were prepared by PyMOL (http://pymol.org/) and present the structural characteristics of class III peroxidases. Colors indicate secondary structures: α-helix (red), β-sheets (yellow), loops (green), disulfides (blue), and haem group (grey).</figcaption></figure>

**4. Topology and Spatiotemporal Organization**

All class III peroxidases appear to have a cleavable signal peptide in common. In silico analyzes of 142 class III peroxidase sequences from maize predicted putative signal peptides, transmembrane domains and the localization of these proteins. This approach revealed a ratio of 53% soluble to 47% membrane-bound isoenzymes [14]. Among the membrane proteins, signals for localization at the endoplasmatic reticulum (ER, 42%), PM (55%), Golgi (2%) and tonoplast (1%) were predicted.

A comparison between rice, barrel medic, arabidopsis and maize showed similar results about the number of isoenzymes with putative transmembrane helices, 51% in rice (n = 78), 49% in maize (n = 77), 35% in barrel medic (n = 36) and 44% in arabidopsis (n = 33). The protein subcellular localization prediction tool (PSORT) indicated a PM localization for at least eight class III peroxidases in arabidopsis, 17 in barrel medic, 13 in rice and 27 in maize (Table 1). Additionally, peroxidases were predicted for ER, Golgi, mitochondria outer (MoM) and inner (MiM) membranes. A vacuolar localization was predicted for seven peroxidases in rice and barrel medic, for six in maize, and for one in arabidopsis. A possible
localization of these vacuolar peroxidases at the tonoplast will need further proof. For maize roots, peroxidases have been detected at the tonoplast [51].

The properties of PM-bound peroxidases from maize and the identification of class III peroxidases in DRM suggested a tight interaction with the membrane [14,22,24,33,34]. According to membrane-bound ascorbate peroxidases that interact with the membrane via a C-terminal membrane-spanning segment [16,52–59], PM-bound class III peroxidases appeared to be anchored by an uncleavable N-terminal signal peptide with a transmembrane helix. Beyond that, proteomic approaches suggest protein-protein interaction [60].

### 4.1. Transmembrane Spanning Domains

In general, N-terminal signal peptides are cleaved off during maturation of soluble secretory peroxidases from eukaryotic organisms. In many cases, the resulting N-terminal positioned glutamine is catalyzed to a cyclic molecule, the so-called pyrrolidone carboxylic acid (PCA) [61]. Prosite predicted such a modification for 32% of the peroxidases shown in Table 1 [62]. Four (OsPrx36, ZmPrx53, ZmPrx78, ZmPrx132) out of 21 vacuolar peroxidases are modified this way potentially. Pyrrolidone carboxylic acid appear absent in CroPrx01 [21] and vacuolar peroxidases from arabidopsis and barrel medic. One third of the putative PM-bound peroxidases showed an N-terminal glutamine that may be modified after cleavage of the signal peptide [21].

Three transmembrane helices (TMH) were predicted for OsPrx95 by different prediction tools [14]. PSORT identified a cleavable signal peptide at the N-terminus of OsPrx95 and predicted a type I membrane protein (TMH 345...361, C-terminus) with a localization at the PM (Table 1). This prediction was in accordance with the identification of the protein in the PM [26]. In contrast, the hypothetical model showed a transmembrane domain at the N-terminus (Figure 2). All other peroxidases from Table 1 have one TMH at the N-terminus that is overlapping with the ER signal peptide. Signal peptides of membrane-bound proteins can remain and, due to their hydrophobicity, function as an N-terminal transmembrane domain. Actual search algorithms may be not able to make exact predictions of this possibility yet. So far, crystal structures of membrane-bound peroxidases have not been published.

In accordance with the biochemical classification and with the identification of ZmPrx01, ZmPrx66, and ZmPrx70 at the PM, five of *n* = 5 prediction programs detected N-terminal signal peptides in their amino acid sequences [22]. PSORT even predicted a non-cleavable signal peptide in ZmPrx70 and a localization of the enzyme at the PM or ER membrane (Table 1). The hypothesis of a transmembrane-spanning domain was supported by experimental data: (i) non of the maize peroxidases could be washed off from the PM by high salt concentrations or other treatments [22,24,63]; (ii) peroxidases were found in DRM of washed PM [14]; (iii) recombinant peroxidases were localized in the yeast membrane after heterologous expression of their full length amino acid sequences [64].

### 4.2. Protein-Protein Interaction

Molecular masses of ZmPrx01 (70 kDa) and pmPOX2a (155 kDa) suggested a putative protein-protein interaction [22,24]. Analyzes of the maize PM proteome by high resolution clear native electrophoresis (hrCNE) pointed to the participation of a PM-bound guaiacol peroxidase to a putative high molecular mass protein complex of 1092 kDa [60]. Interaction of AtPrx64 with PM-bound Casparian strip domain proteins (CASPs) was demonstrated [28]. For tonoplast, interaction of CroPrx01 with arabinogalactan proteins (AGPs) was suggested [35].

Protein-protein interaction may be supported by post-translational modifications (Table 1). Phosphorylation may change the properties of peroxidases and facilitate protein-protein interaction, although this hypothesis has to be verified.

### 4.3. Spatiotemporal Organization

The proteome of PM microdomains (defined by DRM) is involved in signaling and response to biotic and abiotic stress, cellular trafficking, cell wall synthesis and degradation, and metabolism [65,66].
Evidence for a localization of class III peroxidases in DRM has been presented [14,33,34]. As shown in Figure 3, microcompartimentation allows PM-bound peroxidases to co-localize with ROS producing and detoxifying enzymes in the membrane [25]. Thus PM-bound peroxidases may probably not only detoxify H₂O₂ directly at the site of origin to ensure the optimal protection of the membrane, but could also protect specific functional regions of the PM [14,25] and fulfill specific functions in dependence on their substrates.

<figure><img src="image_104.png" /><figcaption>**Figure 3.** Membrane interaction and spatiotemporal organization of CroPrx01, ZmPrx01 and AtPrx64. (a) Soluble CroPrx01 interacts via an arabinogalactan protein (AGP, blue) with the inner surface of the tonoplast [35,67]. For α-3′,4′-anhydrovinblastine (AVLB) synthesis, the complex anchored next to a proton driven alkaloid antiporter (yellow ellipse) [68,69], (b) ZmPrx01 is anchored in the PM by a transmembrane-spanning domain with its active site at the out-side [22]. For cell wall modification, the enzyme co-localized with Rboh (ruby colored) in microdomains. Simultaneously, superoxide dismutase (SOD, green) and ZmPrx01 regulate ROS levels at the PM [14,70]. (c) Soluble AtPrx64 interact with a transmembrane Casparian strip protein (CASP1, orange) at the PM [28,29,71,72]. A dirigent-like protein (EBS1, grey) facilitates co-localization of the complex with RbohF (ruby colored) in sclerenchyma cells [72]. Casparian strip formation is regulated by MYB36 [29]. For peroxidases solvent accessible surface charges are shown: negative (red); neutral (white), positive (blue).</figcaption></figure>

A co-localization of AtPrx64 with respiratory burst oxidase homolog F (RbohF) has been shown [28]. Besides AtPrx64, RbohF, and a dirigent-like protein (Enhanced Suberin 1, ESB1) were recruited by CASPs to assemble the lignin polymerization machinery [29]. It was shown that this process is regulated by the transcription factor MYB36.

The pattern of CroPrx01:GFP-fusion constructs pointed to localization in mircodomains. A co-localization with AGPs was postulated at the tonoplast [35]. Regulation of CroPrx01 functions were discussed in dependence on the specific localization/orientation.

## 5. Function

Functional analysis and biochemical characterization of membrane-bound class III peroxidases are still fragmentary. It is very difficult to identify the exact function(s) of plant peroxidases because of (i) the huge amount of similar isoenzymes, (ii) the broad substrate specificity, (iii) the high number of possible functions, and (iv) the ability of other isoenzymes to compensate the absence of an enzyme in knock-out experiments [11,73]. Progress has been made by molecular biological approaches in combination with biochemical characterization that resolve some of these problems [74].
## 5.1. Molecular Function

Class III peroxidases are involved in production and scavenging of ROS [7,8,75]. Various substrates are oxidized by the peroxidative cycle that play important functions in polymerization of cell wall components, auxin metabolism, and NAD(P)H oxidation via a non-catalytic reaction. Superoxide anion production via this reaction is immediately converted to hydrogen peroxide and molecular oxygen either spontaneously or through superoxide dismutase. The hydroxylic cycle can produce ROS. Both cycles control hydrogen peroxide levels of cells [25]. A function of hydrogen peroxide in redox regulation and signaling has been demonstrated [76,77].

### Substrates

The PM is the first target of a stressor. Large amounts of hydrogen peroxide are produced at the PM due to cellular processes, as a response to stress factors, or to external sources in plant-pathogen interactions [78–81]. This oxidative stress causes lipid peroxidation and changes in membrane permeability [82–84]. PM-bound peroxidases were suggested to be substantially involved in the detoxification of the cell and/or repair of the PM after oxidative damage [25]. They probably play an important role in maintaining the cell functions under different stress conditions [22].

Class III peroxidases oxidize a wide variety of small phenolic compounds, including monolignols, hydroxycinnamic acids, dimeric alkaloids and others [85,86]. For the docking study shown in Table 2, several of these substrates were tested: (i) monolignols such as coniferyl, sinapyl, and *p*-coumaryl alcohols as precursors of lignin [87,88]; (ii) hydroxycinnamic acids like ferulic acid, caffeic acid and *p*-coumaric acid as precursors of suberin [89]; (iii) *L*-3,4-dihydroxyphenylalanine (*L*-DOPA) as precursor of various alkaloids, catecholamines, and melanin [90,91]; and (iv) other substrates like ascorbic acid, indol-3-acetic acid (IAA) and nicotinamide adenine dinucleotides (NAD(P)H). Auxins like IAA have a crucial role in the cell elongation process [92] and can be accumulated in vacuoles [93].

The docking analysis revealed different substrate affinities for the peroxidases in dependence on their localization and biological function. According to their classification, the peroxidases showed low affinity against ascorbic acid (Table 2).

PM-bound peroxidases of arabidopsis, barrel medic or rice (Figure 1), have not been characterized biochemically yet. For AtPrx64, the substrate affinity decreased in the order NADPH > IAA > NADH > *L*-DOPA > cinnamyl alcohol > sinapyl alcohol > ferulic acid (Table 2). Gene expression of AtPrx64 indicates that this peroxidase is crucial to lignin deposition in the Casparian strip of endodermis cell walls in roots [28,29]. This observation correlates with affinities of the hypothetical structure of the enzyme against alcohols such as cinnamyl and sinapyl (Table 2).

The affinity of AtPrx47 decreased from NADPH > IAA > NADH > ferulic acid > cinnamyl alcohol. The docking analyses revealed that *L*-DOPA appears not to be a substrate for AtPrx47. These results supported a function of AtPrx47 in the lignification of vessels [30].

PM-bound MtPrx02 showed a high affinity for substrates related to cell wall modification, such as IAA, ferulic acid, cinnamyl and sinapyl alcohol (Table 2). This observation confirmed experimental data with this peroxidase [94]. Similar results were found for OsPrx95 that showed high affinity with substrates involved in cell wall modification such as IAA and cinnamyl alcohol (Table 2).

Both, CroPrx01 and AtPrx64, revealed similar affinities with *L*-DOPA, whereas the other peroxidases showed no reaction with this substrate. This observation excluded a localization of AtPrx47, MtPrx02, OsPrx95 or ZmPrx01 at the tonoplast or in the vacuole. As mentioned above, the structure of AtPrx64 may need further elucidation and results from the docking study have to be verified by biochemical characterization of the native enzyme and/or recombinant polypeptide. Biochemical differences between native proteins and recombinant polypeptides may be possible due to a distinct degree of glycosylation.
<table><caption>**Table 2.** Docking results of multiple ligand molecules by Firedock (http://bioinfo3d.cs.tau.ac.il/FireDock/). Putative structures of the membrane-bound peroxidases CroPrx01 (5a0g.1.A), AtPrx64 (3hdl.1.A), AtPrx47 (5twt.1.A), MtPrx02 (5twt.1.A), OsPrx95 (3hdl.1A) and ZmPrx01 (3hdl.1.A) were modeled by SWISS-MODEL (https://swissmodel.expasy.org/). Horseradish peroxidase (HRP, 1hch) was used for comparison.</caption><thead><tr><th>Substrate</th><th>CroPrx01</th><th>AtPrx64</th><th>AtPrx47</th><th>MtPrx02</th><th>OsPrx95</th><th>ZmPrx01</th><th>HRP</th></tr></thead><tbody><tr><td>ascorbic acid</td><td>-5.47</td><td>-5.39</td><td>-5.94</td><td>-5.48</td><td>-5.56</td><td>-5.29</td><td>-5.88</td></tr><tr><td>L-DOPA<sup>1</sup></td><td>-8.23</td><td>-8.39</td><td>0.24</td><td>12.62</td><td>7.11</td><td>12.34</td><td>-0.34</td></tr><tr><td>indole acetic acid</td><td>-15.41</td><td>-13.66</td><td>-12.95</td><td>-17.24</td><td>-13.05</td><td>-13.6</td><td>-11.79</td></tr><tr><td>NADH<sup>2</sup></td><td>-9.26</td><td>-8.6</td><td>-12.82</td><td>-12.42</td><td>-13.32</td><td>-9.89</td><td>-6.29</td></tr><tr><td>NADPH<sup>3</sup></td><td>-12.93</td><td>-13.69</td><td>-13.78</td><td>-11.69</td><td>-11.95</td><td>-12.49</td><td>-5.44</td></tr><tr><td>cinnamyl alcohol</td><td>-7.95</td><td>-7.98</td><td>-7.67</td><td>-8.74</td><td>-7.67</td><td>-6.39</td><td>-5.85</td></tr><tr><td>coniferyl alcohol</td><td>-4.56</td><td>-4.33</td><td>-3.77</td><td>-3.73</td><td>-4.85</td><td>-4.54</td><td>-3.81</td></tr><tr><td>sinalpyl alcohol</td><td>-7.31</td><td>-7.88</td><td>-5.23</td><td>-8.02</td><td>-6.51</td><td>-7.26</td><td>-3.35</td></tr><tr><td>ferulic acid</td><td>-6.8</td><td>-7.18</td><td>-8.14</td><td>-9.04</td><td>-6.11</td><td>-5.22</td><td>-6.86</td></tr><tr><td>caffeic acid</td><td>-5.35</td><td>-5.34</td><td>-5.06</td><td>-5.98</td><td>-5.91</td><td>-4.88</td><td>-2.07</td></tr><tr><td>p-coumaric acid</td><td>-4.96</td><td>-5.77</td><td>-5.32</td><td>-5.46</td><td>-6.08</td><td>-6.4</td><td>-5.54</td></tr></tbody></table>

Cinnamyl alcohol is easily oxidized by most of the peroxidases, but coniferyl and sinapyl alcohol seem to be poor substrates for many of these enzymes. Thus even during lignin synthesis, sinapyl alcohol dehydrogenation may be mediated by other phenolic radicals [95,96].

## 5.2. Biological Functions

The expression of peroxidase enzymes is regulated and induced by several stressors, such as pathogens, temperature, nutrient starvation or environmental changes [74]. Regulation of peroxidases is strongly effected by ROS levels in the plants. These ROS levels are linkups to the stressors, related besides the self-regulation of the plant [97].

Although class III peroxidases are highly conserved, the high similarity in protein structure has no simple correlation with the functions of these enzymes. Nevertheless, the structural features of class III peroxidases, like distribution of surface charges, glycosylation patterns or even the regulation of the enzymes, seem to be important in the classification by functions of the enzymes [74]. Another feature that was used to characterize peroxidases is the isoelectric point (pI) [6]. So far, little or no information can be found on the correlation between pI and biological functions.

Both, alkaline and acidic stress, generate an increase of ROS in plant tissues [98–100]. Those changes in soil pH and apoplast have a significant effect on the regulation of peroxidases that are linked to ROS generation and scavenging. On the one hand, several studies showed optima in the range between pH 4.5 to 6.5 for peroxidase activity [101,102]. On the other hand, pH is crucial for the stability of peroxidases [103].

Evaluation of Table 1 revealed several membrane peroxidases that respond to pH in rice (n = 2) and maize (n = 11), but none in arabidopsis or barrel medic. In maize, a localization of these isoenzymes was predicted for PM (n = 7), vacuole (n = 2) and mitochondria inner membrane (n = 2). In rice, two isoenzymes were predicted for PM. All these peroxidases were anionic with the exception of ZmPrx140 (Table 1).

A proteomic approach suggested specific functions of ZmPrx01, ZmPrx66, ZmPrx70 and pmPOX2a in response to oxidative stress [63]. Both, substrate affinity and specific activities make a function of ZmPrx01 in lignification most likely. Several putative cis-regulatory elements were identified by partial gene analysis of ZmPrx70 that suggested a regulation of its gene by wounding, methyl jasmonate, salicylic acid, and elicitors [22]. In addition, a regulation of the gene by oxidative stress was suggested by seven successive elements with sequence similarity to the consensus sequence of antioxidant-responsive elements (ARE) at the 5'-untranslated region of ZmPrx70 [63,104].

<footnote n="1">¹ DOPA, L-3,4-Dihydroxyphenylalanin; ² NADH, Nicotinamide adenine dinucleotide; ³ NADPH, Nicotinamide adenine dinucleotide phosphate.</footnote>
Recent studies showed that high-light conditions can increase phenol and peroxidase levels and by that the hydrogen peroxide scavenging capacity of the vacuole. In fact it was proposed that the vacuolar couple CroPrx01/secondary metabolites represent an important sink/buffer of hydrogen peroxide in green plant cells [105].

### 5.2.1. Orthologous

According to Welinder et al. [61], truly orthologous of peroxidases should have sequence identities greater than 90%. For the peroxidases discussed above, orthologous were not found for the species investigated. Sequence similarities above 70% might indicate related physicochemical properties and physiological functions.

Homologous of AtPrx47 appear not to exist in arabidopsis, highest sequence similarity was 47% (Table 3). For AtPrx47, a function in lignification of vessels has been shown during plant development [29,30]. This observation was supported by the docking analyzes (Table 2). Several proteins with sequence similarities above 80% to AtPrx47 were found in the Brassicaceae family, e.g., AruPrx47 (94.9%) from horseradish (*Armoracia rusticana* G.GAERTN., B.MEY (Bernhard. Meyer). and SCHERB. (Scherbius, Johannes)). Sequence similarities above 70% were indicated for MtPrx19 (G7IJT4) and CroPrx09. However, MtPrx19 was suggested to have a function in response to pathogens, whereas the function of CroPrx09 is unknown. For maize and rice, sequence similarities were below 60%. In contrast to AtPrx47, none of the putative orthologous appear to have a transmembrane-spanning domain or a localization at the PM.

<table><caption>**Table 3.** Homologous and most similar sequences of membrane-bound class III peroxidases in other species. Sequence identity is given in brackets. Protein names are given according to PeroxiBase.</caption><thead><tr><th>Protein</th><th>Z. mays</th><th>O. sativa</th><th>M. truncatula</th><th>A. thaliana</th><th>C. roserus</th></tr></thead><tbody><tr><td>CroPrx01</td><td>ZmPrx16 (55%)</td><td>OsPrx23 (53%)</td><td>MtPrx48 (54%)</td><td>AtPrx12 (59%)</td><td>CroPrx03 (84%)</td></tr><tr><td>AtPrx64</td><td>ZmPrx136 (65%)</td><td>OsPrx32 (60%)</td><td>MtPrx55 (72%)</td><td>AtPrx66 (52%)</td><td>CroPrx40 (72%)</td></tr><tr><td>AtPrx47</td><td>ZmPrx15 (57%)</td><td>OsPrx117 (60%)</td><td>MtPrx19 (71%)</td><td>AtPrx66 (47%)<br>AtPrx64 (46%)</td><td>CroPrx43 (72%)<br>CroPrx09 (75%)</td></tr><tr><td>MtPrx02</td><td>ZmPrx120 (52%)</td><td>OsPrx40 (59%)</td><td>MtPrx70 (96%)</td><td>AtPrx52 (46%)</td><td>CroPrx04 (55%)</td></tr><tr><td>OsPrx95</td><td>ZmPrx94 (73%)</td><td>OsPrx97 (50%)<br>OsPrx134 (50%)</td><td>MtPrx07 (43%)</td><td>AtPrx56 (46%)</td><td>CroPrx49 (48%)<br>CroPrx59 (47%)</td></tr><tr><td>ZmPrx1</td><td>ZmPrx101 (68%)</td><td>OsPrx12 (74%)</td><td>MtPrx07 (52%)</td><td>AtPrx39 (46%)</td><td>CroPrx14 (48%)</td></tr></tbody></table>

Similar results have been found for the tonoplast-bound CroPrx01. Sequence similarity of 84% was found between CroPrx01 and CroPrx03 (B2G335), but in maize, rice, barrel-medic or arabidopsis, sequence similarities were below 60%. CroPrx03 is a soluble apoplastic peroxidase that is expressed in stem and flower tissues [106]. The transcript was down-regulated under salt and dehydration stress.

For the barrel medic MtPrx02, MtPrx70 (G7KFK8) was found as homologous by sequence similarity (Table 3). Indeed this peroxidase responded to nitrogen starvation and pathogen infection. In contrast to MtPrx02, MtPrx70 respond to mycorrhiza and appears to be a soluble peroxidase. PSORT predicted localization outside the cell and the enzyme does not contain a transmembrane helix. Arabidopsis, rice and maize showed similarities below 60% (Table 3).

True orthologous of OsPrx95 were not indicated in maize, barrel medic or arabidopsis. Highest sequence similarity to OsPrx95 was found for ZmPrx94 (A0A1D6HQQ8). Both proteins were predicted at the PM (Table 1). Salt stress caused a decrease in OsPrx95 abundance [27]. In contrast, ZmPrx94 was induced by salinity. The lignin biosynthesis pathway altered significantly under salt stress. For example, cell wall thickness of vessels in sorghum [107] and thickness of Casparian strip in maize roots [108] increased during acclimation to salinity.

Orthologous of ZmPrx01 were not found in rice, Madagascar periwinkle, barrel medic or arabidopsis. In maize, ZmPx101 (B4FU88) and ZmPrx49 (B4FY83) showed highest sequence similarity
to ZmPrx01. Both proteins appear to be soluble peroxidases. ZmPrx101 is induced by pathogens, whereas the function of ZmPrx49 is unknown.

Similar results were found by comparison of all ER, PM and vacuolar peroxidases from arabidopsis, barrel medic, rice and maize. Among ER-bound peroxidases, the highest sequence identity was between ZmPrx135 and MtPrx58 (47.33%), the lowest between ZmPrx46 and AtPrx69 (37.15%). In total, 45 putative PM-bound peroxidases were found. The highest sequence identities were found for ZmPrx41 and ZmPrx55 (97.95%) > ZmPrx66 and ZmPrx114 (80.89%) > OsPrx135 and ZmPrx115 (80.43%) > OsPrx30 and ZmPrx123 (79.88%), but for most of them sequence identities were below 60%.

ZmPrx41 responded to pH, drought and etiolation, whereas ZmPrx55 appears to have a function in cold stress. ZmPrx66 showed response to drought, ZmPrx114 appears to have a function in drought stress, but responded also to pathogens and pH (Table 1). ZmPrx115 was affected by etiolation, and is involved in cell wall modification whereas the function of OsPrx135 is unknown.

For vacuolar peroxidases sequence identities were below 60% with the exception of ZmPrx53 and ZmPrx132 (86.28%). Functions of these peroxidases are unknown.

### 5.2.2. Phylogeny

A phylogenetic tree was performed to obtain further clues to the biological functions of membrane-bound peroxidases in maize, rice, barrel medic and arabidopsis (Figure 4). The phylogenetic tree was constructed using the maximum likelihood method based on the Jones, Taylor, Thornton (JTT) matrix-based model [99]. The analysis was done with amino acid sequences of peroxidases from Table 1. In total 110 sequences were analyzed, Eleven from arabidopsis, 31 from barrel medic, 22 from rice, 45 from maize and one from the liverwort *Marchantia polymorpha* (MpPrx92). The tree was rooted to MpPrx92. The amino acid sequence reveals no signal peptide, it has a prediction for 1 TMH and for a localization of the protein at the PM. Phylogenetic analysis allows identifying the evolutionary conservation and divergence of these enzymes from the common ancestor MpPrx92.

The tree was divided into six major groups (A to F). The first group, which clusters peroxidases that respond to various stresses, is subdivided in four clusters (A1 to A4). Cluster A1 summarized peroxidases that were characterized to work against several stresses, like drought (e.g., MtPrx29, ZmPrx66 and ZmPrx70), cold (ZmPrx55), pathogens (e.g., MtPrx02, MtPrx32 and ZmPrx86) and even hormones (e.g., AtPrx10 and MtPrx94). Cluster A2 contains only two PM peroxidases that participate in oxidative stress (OsPrx117) and lignification of vessels (AtPrx47). In fact the similarity between the two peroxidases is remarkable (Table 3), but the similarities of biological functions need further proofs. In group A3, peroxidases clustered together that are involved in reactions against pathogens (e.g., AtPrx64, MtPrx55 and MtPrx92) and nodulation (e.g., MtPrx13, MtPrx55 and MtPrx92). Nodulation is characteristic for legumes like barrel medic [109]. Peroxidases from other species in this cluster work most probably in defense against pathogens. Finally, the cluster A4 is formed exclusively by PM peroxidases of maize that are involved against stresses like drought (ZmPrx81) and defense against pathogens (ZmPrx85).

Group B is subdivided into two clusters but there is not so much known about this group.

Group C is subdivided in five clusters with different responses to abiotic stress. Group C1 contains peroxidases that were not characterized experimentally. A BLAST search suggests a function in response to pH changes, such as soil acidification. This group is formed only by maize peroxidases, with the exception of OsPrx95 from rice. Maize and rice are two of the bigger crops in the world, and frequently the growing of these plants is related to soil conditions. Low pH causes less growing of the plant and even death. So it is very interesting to find peroxidases in these two species that might be involved in this kind of stress. Additionally, this stress can be related to other stresses, like nutrient starvation or drought [112]. The C2 cluster is build by two PM peroxidases that are involved in plant development. AtPrx18 is related to the development of the floral organ and OsPrx5556 could be involved in the response to hormones and heat.
<figure><img src="image_105.png" /><figcaption>**Figure 4.** Phylogenetic analysis of peroxidases from Table 1 and MpPrx92 by the maximum likelihood method. The evolutionary history was inferred by using the maximum likelihood method based on the Jones, Taylor, Thornton (JTT) matrix-based model [110]. The tree with the highest log likelihood (-58984.24) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 110 amino acid sequences. There were a total of 841 positions in the final dataset. Evolutionary analyses were conducted by Molecular Evolutionary Genetics Analysis (MEGA7) [111]. ●, ER; ●, PM; ●, Vac; ■, Golgi; ▲, MoM; ▼, MiM.</figcaption></figure>
Peroxidases from group C3 seem to be involved in nitrogen starvation response. Three peroxidases of three different species, MtPrx04, OsPrx138 and ZmPrx72, build this cluster and, despite the fact that only one of these peroxidases was studied (MtPrx04), the similarity between them is so high that they may have the same function. This could mean that these peroxidases have a common origin to protect the plant against nutrient deficiency. Cluster C4 is build by peroxidases that are involved in processes like cell wall modification (e.g., ZmPrx01). Finally, group C5 contains peroxidases that are not characterized. A BLAST search suggested a function in response to different abiotic stresses like pH and drought (ZmPrx78) or nitrogen and phosphate starvation (OsPrx46 and OsPrx107).

Group D is formed by three peroxidases from barrel medic and rice that are not characterized.

Group E include mainly peroxidases that are involved in etiolation and are regulated by light and dark (e.g., ZmPrx104 and ZmPrx112). However, these functions are only theoretical because none of these peroxidases were characterized experimentally.

Finally, group F is involved mainly in nodulation (e.g., MtPrx41, MtPrx42 and MtPrx74). In accordance with this specialization of legumes, the majority of peroxidases were from barrel medic. Two peroxidases were from maize (ZmPrx57 and ZmPrx71), and these were separated from the others. The genes showed distinct expression patterns: *zmprx57* has been found in immature leaf, whereas *zmprx71* has been expressed in primary root [113]. Both proteins have a prediction for PM localization. At least one of them may respond in defense against pathogens. This function can be easily related to the nodulation in barrel medic plants. However, biochemical characterization of the maize peroxidases is still missing.

## 6. Conclusions

Molecular biodiversity of membrane-bound class III peroxidases appears to be higher than expected. Although plants evolved the same molecular mechanisms, true orthologues of membrane-bound peroxidases were not identified in the four species investigated. Besides soluble class III peroxidases that interact with the membrane by protein-protein interaction, type I membrane proteins may exist. Membrane-bound peroxidases are multifunctional enzymes that fulfill essential functions in plant development and stress response. Microcompartmentation and co-localization of PM-bound peroxidases with ROS-producing and detoxifying enzymes may probably not only detoxify hydrogen peroxide directly at the site of origin, but could also protect specific functional regions of the PM and fulfill specific functions. Biochemical characterization of membrane-bound peroxidases is still fragmentary and will need further elucidation. Some of these peroxidases may have the potential for marker-assisted breeding.

**Author Contributions:** S.L. designed the concept and structure of the paper, she prepared Figures 1–3, Tables 1 and 3, and wrote the main text. T.M.-C. performed the docking analyzes and the phylogenetic tree, prepared Figure 4, Table 2 and wrote the interpretation of phylogenetics. S.L. and T.M.-C. discussed and corrected the manuscript.

**Acknowledgments:** Financial support was given to S.L. by a research grant from DFG (DFG Lu-668/4-4) and to T.M.C. by a post doctoral grant (ED481B 2016/221-0) from the Xunta de Galicia, Spain. Funds for covering the costs to publish in open access were not available.

**Conflicts of Interest:** The authors declare no conflict of interest.

## Abbreviations

AGP Arabinogalactan protein

ARE Antioxidant-responsive elements

AtPrx47 Arabidopsis thaliana peroxidase 47

AtPrx64 AtPer64, Arabidopsis thaliana peroxidase 64

AVLB -3',4'-anhydrovinblastine

BLAST Basic local alignment search tool

BvPrx12 Beta vulgaris peroxidase 12

CASPs Casparian strip domain proteins
<table><tr><td>CroPrx01</td><td>CrPrx1; Catharanthus roseus peroxidase 1</td></tr><tr><td>DRM</td><td>Detergent resistant membrane</td></tr><tr><td>ER</td><td>Endoplasmic reticulum</td></tr><tr><td>ESB1</td><td>Enhanced Suberin 1</td></tr><tr><td>GAERTN.</td><td>Gärtner, Joseph</td></tr><tr><td>GFP</td><td>Green fluorescence protein</td></tr><tr><td>hrCNE</td><td>High resolution clear native electrophoresis</td></tr><tr><td>HEYNH.</td><td>Heynhold, Gustav,</td></tr><tr><td>HRP</td><td>Horseradish peroxidase</td></tr><tr><td>IAA</td><td>Indol-3-acetic acid</td></tr><tr><td>L-DOPA</td><td>L-3,4-dihydroxyphenylalanine</td></tr><tr><td>MEGA7</td><td>Molecular Evolutionary Genetics Analysis Version 7.0</td></tr><tr><td>B.MAY.</td><td>Bernhard. Meyer</td></tr><tr><td>MiM</td><td>Mitochondrial inner membrane</td></tr><tr><td>MoM</td><td>Mitochondrial outer membrane</td></tr><tr><td>MS</td><td>Mass spectrometry</td></tr><tr><td>MtPrx02</td><td>Medicago truncatula peroxidase 2</td></tr><tr><td>MW</td><td>Molecular weight</td></tr><tr><td>MYB36</td><td>MYB domain protein 36</td></tr><tr><td>NADH</td><td>Nicotinamide adenine dinucleotide</td></tr><tr><td>NADPH</td><td>Nicotinamide adenine dinucleotide phosphate</td></tr><tr><td>OsPrx95</td><td>Oryza sativa peroxidase 95</td></tr><tr><td>PCA</td><td>Pyrrolidone carboxylic acid</td></tr><tr><td>pI</td><td>Point isoelectric</td></tr><tr><td>PM</td><td>Plasma membrane</td></tr><tr><td>pmPOX2a</td><td>Plasma membrane peroxidase 2a</td></tr><tr><td>PSORT</td><td>protein subcellular localization prediction tool</td></tr><tr><td>PsPrx13</td><td>Pisum sativum peroxidase 13</td></tr><tr><td>PyMOL</td><td>Python-enhanced molecular graphics tool</td></tr><tr><td>Rboh</td><td>Respiratory burst oxidase homolouge</td></tr><tr><td>ROS</td><td>Reactive oxygen species</td></tr><tr><td>SCHERB.</td><td>Scherbius, Johannes</td></tr><tr><td>SOD</td><td>Superoxide dismutase</td></tr><tr><td>TMH</td><td>Transmembrane helix</td></tr><tr><td>TMHMM</td><td>Trans Membrane Helix Markov Model</td></tr><tr><td>Vac</td><td>Vacuole</td></tr><tr><td>ZmPrx01</td><td>Zea mays peroxidase 1</td></tr></table>

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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Review

# The Multifaceted Role of Pectin Methylesterase Inhibitors (PMEIs)

Alexandra Wormit<sup>1,2,*</sup> and Björn Usadel<sup>1,2,3</sup>

Received: 5 August 2018; Accepted: 5 September 2018; Published: 21 September 2018

**Abstract:** Plant cell walls are complex and dynamic structures that play important roles in growth and development, as well as in response to stresses. Pectin is a major polysaccharide of cell walls rich in galacturonic acid (GalA). Homogalacturonan (HG) is considered the most abundant pectic polymer in plant cell walls and is partially methylesterified at the C6 atom of galacturonic acid. Its degree (and pattern) of methylation (DM) has been shown to affect biomechanical properties of the cell wall by making pectin susceptible for enzymatic de-polymerization and enabling gel formation. Pectin methylesterases (PMEs) catalyze the removal of methyl-groups from the HG backbone and their activity is modulated by a family of proteinaceous inhibitors known as pectin methylesterase inhibitors (PMEIs). As such, the interplay between PME and PMEI can be considered as a determinant of cell adhesion, cell wall porosity and elasticity, as well as a source of signaling molecules released upon cell wall stress. This review aims to highlight recent updates in our understanding of the PMEI gene family, their regulation and structure, interaction with PMEs, as well as their function in response to stress and during development.

**Keywords:** pectin methylesterase inhibitor (PMEI), pectin; homogalacturonan (HG); cell wall properties; degree of methylesterification (DM); stress; development; applications

## 1. Introduction

Plant cells are surrounded by a wall composed of interacting networks of polysaccharides, highly glycosylated proteins and other polymers. Plant cell walls are complex and highly dynamic structures, responding and adapting to normal processes of growth and development as well as to biotic and abiotic stresses. They have to fulfil several different functions: on one hand, they need to be flexible to allow fast and directional cell elongation (e.g., pollen tubes) and on the other hand they have to be rigid enough to resist the internal turgor pressure, to protect against pathogens and to provide structural and mechanical support for the upright growth of the whole organism.

The major carbohydrates of primary cell walls are cellulose, hemicellulose, and pectins. Cellulose and hemicellulose form a hydrogen-bonded network that is embedded in a gel-like pectic matrix. Pectin is a galacturonan-based polysaccharide containing five distinct subclasses that can be distinguished based on the structure of their backbones and the diversity of their side chains: homogalacturonan (HG), rhamnogalacturonan I and rhamnogalacturonan II (RG-II), xylogalacturonan and apiogalacturonan [1]. In dicots and non-graminaceous plants, pectins constitute about 35% of primary cell walls, whereas in grasses only 2–10% of primary walls are pectic polysaccharides [2]. Pectin has been shown to play roles controlling cell wall porosity [3], cell elongation [4], and cell adhesion [5] and constitutes an important factor in plant development (for reviews see [6,7]).

<footnote n="1">¹ Institute of Biology 1, Botany and Molecular Genetics, RWTH Aachen University, 52074 Aachen, Germany; usadel@bio1.rwth-aachen.de</footnote>

<footnote n="2">² Bioeconomy Science Center (BioSC), Forschungszentrum Jülich, Wilhelm Johnen Straße, 52425 Jülich, Germany</footnote>

<footnote n="3">³ Institute for Bio- and Geosciences (IBG-2: Plant Sciences), Forschungszentrum Jülich, 52428 Jülich, Germany</footnote>

<footnote>* Correspondence: awormit@bio1.rwth-aachen.de; Tel.: +49-241-80-26766</footnote>
HG constitutes ~65% of pectin and is the most abundant pectic polysaccharide in primary cell walls [1]. It consists of an α-1,4-linked D-galacturonic acid (GalA) backbone, which is synthesized by HG galacturonosyl-transferases (GAUTs) from the nucleotide sugar uridine diphosphate (UDP)-GalA in the Golgi apparatus. So far, the galacturonosyl-transferase GAUT1 has been biochemically characterized in *Arabidopsis* [8,9] and a hetero-complex formation of two GAUT1 with one GAUT7 molecule has been shown responsible for retaining GAUT1 in the Golgi apparatus [10]. In addition, GAUT4 from *Arabidopsis thaliana*, switchgrass and poplar was shown to synthesize HG and downregulate of the gene reduced HG and RG-II in the cell wall [11]. When HG is secreted into the cell wall, it is highly methyl-esterified at the C6 atom of the GalA residue (~80% [2]) and a putative pectin methyltransferase conferring this methyl-esterification has been identified in the Golgi [12,13]. In addition, some GalA residues of HG carry acetyl groups at O2 or O3, which affect the physiochemical properties of HG in vitro [14].

Once incorporated into the cell wall, HG is further selectively modified and the pattern and degree of these modifications affect pectin hydrolysis and properties such as pH, charge and crosslinking. Pectin acetyl esterases catalyze de-acetylation and the release of acetate from the cell wall, causing changes in cell wall mechanics and developmental aberrations [15,16]. Pectin methyl esterases (PMEs) catalyze the specific de-methylesterification of HG, releasing methanol and protons, and creating negatively charged carboxyl groups in the process (Figure 1). The degree and pattern of methylesterification of HG determine the biomechanical properties of the cell wall. When several consecutive GalA residues are de-methylesterified (block-wise de-methylesterification), the negatively charged carboxyl groups can form calcium bonds with other HG molecules, leading to so-called ‘egg-box’ structures that underlie the formation of pectin gels [17]. De-methylesterified, calcium cross-linked HG increased the amount of bound water maintaining wall hydration [18] and the hydration state was shown to affect biomechanical properties of the cell wall, such as its rigidity [19]. In addition, the strength of pectin gels is highly dependent on the amount of free calcium ions in the apoplast, as stiffness of the gel is reduced by disassociation of calcium crosslinks [20]. On the other hand, partially de-methylesterified HG (random or block-wise de-methylesterification) can become a target for pectin-degrading enzymes such as polygalacturonases and pectate/pectin lyases.

The degree of methylesterification (DM) of HG, which is controlled by the activity of large PME families, has vast consequences on the mechanical properties of the cell wall [21], affecting developmental processes such as stomata opening [22,23], cell adhesion [5], organ initiation [24] and anisotropic cell growth [25]. In addition, hydrolysis of partially de-methylesterified HG can lead to the formation of signaling molecules (oligogalacturonides), for instance, during plant-pathogen interactions [26,27]. Consequently, PME activity is tightly regulated at: (a) the transcriptional level [28], (b) by protein processing [1] and degradation [29], (c) by the pH of the cell wall environment [6,30], and (d) by endogenous inhibitor proteins called pectin methylesterase inhibitors (PMEI, Figure 1) [31,32]. The first PMEI was identified in kiwi fruit (*Actinidia deliciosa* [33]) and to date several PMEIs have been investigated in different plant species. This review therefore aims to give a comprehensive overview on the current knowledge about PMEIs and their various roles in plant development and stress response.
<figure><img src="image_108.png" /><figcaption>**Figure 1.** Schematic diagram showing the de-methylesterification of HG and the effects on its structure. HG is highly methylesterified when deposited into the cell wall. PMEs can de-methylesterify HG in a block-wise fashion, leading to several consecutive GalA residues without methylester groups. These HG backbones are negatively charged and can therefore form crosslinks with cations like calcium ions, leading to so called ‘egg-box’ structures responsible for gel formation. On the other hand, PMEs can de-methylesterify single GalA residues leading to a random methylesterification pattern. Low-methylesterified HG is depolymerized by pectin-degrading enzymes such as polygalacturonases (PG) and pectin/pectate lyases (PL), which leads to the formation of oligogalacturonides (OG). PME activity is inhibited by its proteinaceous inhibitor PMEI.</figcaption></figure>

## 2. PMEI Occurrence and Regulation

PMEIs belong to large multigene families, containing almost as many members as *PME* genes in several plant species [34]. The PMEI proteins first appeared in mosses (*Physcomitrella patens*), which coincided with the appearance of pectin in cell walls [34]. In *Arabidopsis*, 71 putative PMEI genes have been identified in silico (not including *proPME* genes containing PMEI domains) [34], compared to 100 open reading frames (ORFs) in *Brassica campestris* [35] and 97 putative PMEI genes in *Brassica rapa* [36]. Pinzón-Latorre and Deyholos [37] listed 95 PMEI ORFs in flax (*Linum usitatissimum*), whereas in poplar (*Populus trichocarpa*), only 54 genes have been annotated [34]. In monocots, the PMEI families generally contain fewer isoforms. For example, in rice (*Oryza sativa*), 49 ORFs were annotated as putative PMEIs [38] compared to 37 genes in *Sorghum bicolor* [34] and 38 putative PMEI members in *Brachypodium distachyon* [1]. The smaller family size in grasses is likely due to differences in the structure of cell wall polysaccharides, with pectins being less abundant and less methylesterified in graminaceous species [2,28]. Gene family synteny and phylogenetic analyses suggest that the expansion of the PMEI families in several angiosperm species is due to whole genome duplication and tandem duplication events during evolution [34,35,37].

PMEIs are crucial factors in regulating the DM of HG, which has tremendous effects on cell wall mechanics and affects many biological processes (Figure 2).
<figure><img src="image_109.png" /><figcaption>**Figure 2.** Effect of PMEI regulation on cell wall properties and biological processes affected by PMEI manipulation. PMEIs are transcriptionally regulated in a tissue-specific and development-dependent manner. Several plant hormones and signaling molecules as well as environmental stresses can activate PMEI gene expression. Alternative splicing and directed endocytosis and secretion regulate the level of active PMEIs in the cell wall. PMEI can inhibit several PME enzymes, thereby regulating de-methylesterification of HG. This in turn modulates cell wall properties such as loosening or strengthening, which is required for several biological processes.</figcaption></figure>

Therefore, it is not surprising that their expression is highly coordinated with other pectin modifying enzymes, such as PMEs [28]. Transcriptional analyses (using microarrays, RNAseq, qRT-PCR) showed that PMEI expression is spatially and developmentally controlled in various species (Brassica rapa [36], Brassica campestris [39], Arabidopsis [40], grapevine [41], wheat [42], rice [38], and flax [37,43]). Cluster analysis for a fraction of PME and PMEI genes already identified expression clusters specific for e.g., seed coat, endosperm, pollen, shoot apex in *Arabidopsis* [1]. For example, *AtPMEI6* and *AtPMEI14* are specifically expressed in seed coat epidermal cells and their expression is modulated by the transcriptional regulators GLABRA2 and MUCILAGE-MODIFIED 1 (MUM1), known to be involved in seed coat differentiation and mucilage production [44]. In addition, the R2R3-MYB (*myeloblastosis*) transcription factor MYB52 has been identified as a negative regulator of pectin de-methylesterification in seed coat mucilage due to its control of *AtPMEI6* and *AtPMEI14* expression [45].

Hormones are also involved in regulation of PMEI expression in several species [38,46–49]. For example, transcriptional activation of PMEI was shown to be ethylene-dependent during the ripening process of banana fruits [46], whereas a wheat PMEI was inducible by salicylic and jasmonic acid as well as hydrogen peroxide, indicating a role in defense responses [48]. Moreover, post-transcriptional regulation has also been indicated for PMEIs. Rocchi et al. [42] showed that two PMEI genes from durum wheat undergo intron retention. Mature transcripts were predominantly found in floral organs, indicating a role in flower development and in particular, anther and pollen development.

In pollen tubes, dynamic pectin metabolism is the key to polar growth and concurrently, different PME and PMEI isoforms are highly expressed in this cell type. By immuno-labelling, it was shown that HG with a low DM is found at the flanks of the pollen tube, whereas methylesterified pectin is predominantly present at the growing tip region where wall extensibility is required for
cell growth [50,51]. This transition from apical to distal pectin epitopes seems to be correlated with an increase in cell wall rigidity and decrease in visco-elasticity. The maintenance of the distribution of esterified and de-esterified HG is ensured by a dedicated mechanism involving localized secretion of specific PMEI proteins [52]. The pectin methylesterase AtPPME1 is distributed evenly in the cell wall of the pollen tube. In contrast, the pollen-specific AtPMEI2, which interacts with AtPPME1, is exclusively localized at the cell wall of the pollen tube tip. This specific localization was shown to be dependent on endocytic internalization of AtPMEI2 at the flanks of the pollen tube. Therefore, protein secretion and endocytosis seems to be an efficient mechanism for regulating DM of pectin in a temporally and spatially controlled manner in pollen tubes. It remains to be seen if other PMEIs in other plant tissues are also regulated by such a mechanism.

In general, the large number of PMEI family members with distinct expression and regulation patterns could suggest the existence of dedicated interaction pairs required for localized modifications of HG methylesterification during development. Additionally, this could indicate different inhibitory activities of PMEIs depending on the cell wall environment and/or different specificities for target PMEs to guarantee a development- and stress-dependent adjustment of cell wall mechanics.

### 3. PME Inhibitor Structure and Interaction with Pectin Esterases

PME inhibitors and invertase inhibitors belong to the same protein family (PF04043, http://pfam.xfam.org/family/PF04043), although their respective target proteins are part of different metabolite pathways. The overall sequence identity between PMEIs and invertase inhibitors is only moderate (20–30%), but their three-dimensional structure is highly similar [53]. As shown by crystallographic approaches, PMEIs consist of four long α-helices arranged in an up-down-up-down topology, forming a four-helix bundle. They contain an N-terminal signal peptide responsible for extracellular targeting and four highly conserved cysteine residues, which are involved in the formation of two disulfide (S-S) bridges essential for the maintenance of the secondary structure of the protein [54,55]. One S-S bridge connects helices α2 and α3; the second S-S bridge is located in a fifth, N-terminal anchor region consisting of short distorted helices [56].

Interaction studies performed in vitro at different pH values (pH 5.5, 7.5 & 8.5; pH 7.5; pH 5.5 & 7.5; pH 6.5, 7.5 & 8.5) indicated formation of a stoichiometric 1:1 complex of PMEIs with endogenous PMEs but also with enzymes from other species [41,53,56,57]. For example, Jolie et al. [58] showed that purified PMEI from kiwi can bind to PMEs from carrot and broccoli (at pH 6.5), whereas tomato and *Arabidopsis* PMEIs can interact and inhibit PMEs from orange (at pH 7.5 and pH 7, respectively) [30,59]. On the other hand, plant PMEIs do not seem capable of inhibiting fungal or bacterial PMEs, as was shown for PMEI1 and PMEI2 from *Arabidopsis*, which did not affect PME activity of *Botrytis cinerea* [26]. Similarly, Reca et al. [59] showed that purified tomato PMEI only inhibited plant PMEs but was not active against PME from *Erwinia chrysantemi*. An explanation for this lack of inhibition might be that residues critical for PME-PMEI interaction are not conserved in fungal and bacterial PMEs [56].

The three-dimensional structure of *Arabidopsis* and kiwi PMEI interacting with purified PME from tomato revealed that the inhibitor covers the putative active site of the enzyme, thus preventing substrate binding [53]. The N-terminal anchor region interacts with the C-terminus of PME, potentially supporting the structural stability. The complex formation of PMEI with PME has been shown to be pH sensitive [60]. A detailed study on kiwi PMEI indicated that PMEI-PME complex formation in vitro was reduced at pH values above 6.0, due to reversible conformation changes in the 3D structure of the PMEI protein. At pH levels above 7.5, irreversible changes in PMEI secondary structure occur, such as cleavage of the S-S bridges, inducing helix instability. Once the PMEI-PME complex is formed, it is rather stable and only dissociates under extreme pH conditions (such as pH 10), indicating that interaction with the endogenous PME enzyme protects the inhibitor from conformational changes. Different PMEIs have been shown to function at different pH values; Nguyen et al. [61] analyzed a PMEI of rice (OsPMEI28), which had a highest inhibitor activity against commercial PME at pH 8.5, whereas
complex formation between PME3 and PMEI7 of *Arabidopsis* was highly sensitive to pH variation between 5 and 7 [62]. The observed sensitivity was the result of changes in the protonation of amino acid residues at the interface of the two proteins, which caused residues to shift between inter- and intra-molecular interactions. These results indicate that the prevalent cell wall environment, i.e., the pH of the cell wall, can affect the activity of PMEIs, thereby fine-tuning the DM of pectic polysaccharides.

It was also shown that one PMEI protein can bind and inhibit several endogenous PME enzymes [39,56], indicating a complex mechanism for regulation of PME activity. For example, key residues involved in the interaction between the inhibitors AtPMEI4 or AtPMEI9 and AtPME3 were identified by computational approaches and indicated that functional diversity between the two PMEIs leads to distinct consequences on pollen tube elongation [63]. However, the existence of strictly specific PME-I-PME pairs cannot be excluded at this time.

## 4. Role of PMEIs in Stress

The cell wall is the first barrier against pathogens and maintenance of cell wall integrity plays an important role in pathogen defense [64]. For many bacteria and fungi, secretion of cell wall degrading enzymes that hydrolyze pectin is an important component for successfully infecting a plant [65-68]. Conversely, inactivation of pectin degrading enzymes in pathogens can reduce their virulence on host plants [69-72]. Plant PME activity and the level of pectin methylesterification are highly regulated by pathogens during an infection process and an increase in PME activity is correlated with reduced DM of pectin in *Arabidopsis* during infection with different pathogens [67,68,73]. In addition, degradation of HG by pathogenic polygalacturonases releases de-esterified pectin fragments or oligogalacturonides (OGs) which in turn act as damage-associated molecular patterns (DAMPS [74]). Several studies demonstrated that OGs are sensed by specific plant receptors (wall-associated kinase, WAK) and can activate a stress response by the plant [75,76]. It has been shown that the DM of OGs influences the strength of the elicited response [27,77]. Furthermore, PME activity on HG results in release of methanol, a signaling molecule that is involved in priming of (neighboring) plants and able to retard bacterial growth [78].

Constitutive expression of the *PMEI* genes *AtPMEI-1* and *AtPMEI-2* in *Arabidopsis* reduced the PME activity and resulted in an increase in DM of HG [26]. The transgenic mutants were more resistant to the necrotrophic fungus *Botrytis cinerea*, which was not due to inhibition of fungal PME but rather to an impaired fungal growth on methylesterified pectin. Further *PMEI* genes, *AtPMEI10*, *AtPMEI11* and *AtPMEI12*, were identified as upregulated in response to *B. cinerea* infection [47]. Genetic studies indicated that AtPMEI expression is dependent on jasmonic acid and ethylene signaling but only AtPMEI11 can be induced by OGs [47]. The *pmei10*, *pmei11* and *pmei12* mutants showed increased PME activity, decreased DM of pectin and increased lesion formation upon *B. cinerea* infection. This indicates that plants modulate PME activity by expressing PMEIs in response to pathogen attack.

Similarly, transgenic wheat lines expressing the *PMEI* from kiwi fruit also showed a reduction of disease symptoms caused by two fungal pathogens, *Fusarium gramineum* and *Bipolaris sorokiniana* [79]. This was related to reduced fungal growth on pectin with high DM and the activity of fungal PG to hydrolyze the plant pectin was impaired. In addition, the transgenic wheat plants showed less disease symptoms upon infection with the biotrophic fungal pathogen *Claviceps purpurea* [80]. This also shows the importance of pectin (and its methylesterification status) in grass species, despite its low abundance [2]. In barley (*Hordeum vulgare*), a family of putative pectin esterase inhibitors (PEIs) has been associated with a resistance gene (*Rrs2* locus), which is involved in defense against *Rhynchosporium commune* which causes leaf blotch [81]. Overexpression of *HvPEI4* led to an improvement of the resistance, but none of the candidate genes alone caused a high increase in resistance level. In a recent study, a PME inhibitor from cotton (*Gossypium hirsutum*) was identified as being involved in the defense response to the fungus *Verticilium dahliae* [39]. *GhPMEI3* expression in *Arabidopsis* led to reduced disease symptoms upon infection with *V. dahlia* and silencing of *GhPMEI3* in cotton resulted in an increased susceptibility to the fungus.
Based on current evidence, the proposed role of PMEI in the immune response is the dynamic modulation of the PME activity (Figure 3). The degree of methylesterification determines the susceptibility of the plant cell wall to the pectin-degrading enzymes of the pathogen [82]. Plant PME activity generates methanol as an alarm signal of the damaged self that can regulate the transcription of pathogen-related PMEI genes either directly (e.g., *AtPMEI11* [47]) or via modulation of DAMP or defense hormone signaling (*AtPMEI10*, *AtPMEI11*, *AtPMEI12* [47]). OGs are released by partial hydrolysis of HG by fungal/bacterial polygalacturonases. They serve as DAMPs and bind to WAKs at the plasmamembrane, leading to activation of plant immune signaling, which induces classic defense responses. The DM of OGs can influence the intensity of the defense response. PMEIs (e.g., *AtPMEI10*, *AtPMEI11*, *AtPMEI12* [47]) are transcriptionally upregulated by defense-related signaling pathways including jasmonic acid and ethylene, and inhibit plant PMEs leading to a higher DM of pectin, which makes it less susceptible to being broken down by fungal cell wall degrading enzymes.

<figure><img src="image_110.png" /><figcaption>**Figure 3.** PMEIs modulate PME activity and DM of pectin in response to fungal and bacterial attack. Bacteria and fungi secrete pectin degrading enzymes, like polygalacturonases and pectate/pectin lyases, upon infection. PME activity leads to release of methanol, which serves as an alarm signal, activating the expression of pathogen-related PMEIs. Similarly, OGs, which are generated by degradation of pectin by fungal/bacterial PGs and PLs, serve as DAMPs, leading also to activation of defense related signaling pathways, which have been shown to activate PMEI gene expression. PMEIs inhibit PME activity leading to a higher degree of methylesterification of pectin, which contributes to resistance to fungal/bacterial enzymes resulting in less disease symptoms.</figcaption></figure>

PMEIs have also been implicated to play a role in defense against bacteria. *CaPMEI1* from pepper (*Capsicum annuum*) was identified in pepper leaves infected with *Xanthomonas campestris* pv. *vesicatoria* (*Xcv*) [83]. *Xcv* secretes plant cell wall degrading enzymes like pectate lyases and polygalacturonases upon infection. *CaPMEI1* was transcriptionally upregulated by different biotic stress signals (e.g., salicylic acid, jasmonic acid, ethylene, H₂O₂) and consequently, silenced pepper plants exhibited an increased susceptibility to *Xcv*. Conversely, overexpression of *CaPMEI1* in *Arabidopsis* conferred an enhanced resistance to *Pseudomonas syringae* but not to the biotrophic fungus *Hyaloperonospora parasitica*.
Little is known about the role of PMEIs in viral infections. In tobacco, plant PMEs have been shown to be involved in tobacco mosaic virus movement between host cells via interaction with the viral movement protein [84]. Accordingly, overexpression of PMEIs in tobacco and *Arabidopsis* counteracted the action of PMEs, leading to increased resistance to tobacco mosaic virus and turnip vein clearing virus infection respectively [85]. This was characterized by the reduced or delayed movement of the viruses and decreased disease symptoms. It is suggested that *PMEI* reduces viral spreading via limiting viral-induced PME activity and by hindering the enlargement of plasmodesmata.

Plants respond to abiotic stresses, e.g., water deficit, salt stress, and temperature extremes, with changes in cell wall architecture, although available literature is more focused on transcriptomic data rather than biochemical experiments [86,87]. PMEIs are also involved in the plants' response to changing environmental conditions, however; only little is known about their molecular role. *CaPMEI1* from pepper is transcriptionally induced by cold treatment, drought stress, as well as the plant stress hormone abscisic acid and hydrogen peroxide [83]. *Arabidopsis* lines overexpressing *CaPMEI1* exhibited an increased tolerance to water stress, as shown by improved germination rate and seedling root growth in the presence of high mannitol concentrations as compared to control plants. In addition, a reduced transpiration rate was accompanied by less withering upon drought stress, and improved tolerance to oxidative stress caused by methyl viologen [83].

Several studies using expression analysis have revealed the functional importance of PMEIs in response to abiotic stresses. RT-PCR analysis of the wheat *TaPMEI* showed the responsiveness of the gene to H₂O₂ stimulus, salinity, water stress (caused by polyethylene glycol) and abscisic acid treatment [48]. Time course analysis further indicated a specificity of the transcriptional response based on the investigated tissue (leaf, stem and root). In silico meta expression analysis using Affymetrics array data for rice [38] revealed the importance for several *PMEI* family members in diverse stress responses. Drought stress, salt stress, cold and anaerobic conditions (during germination) were analyzed, and showed specific transcriptional regulations of *PMEI* genes [38]. Similarly, in *Arabidopsis*, the effect of hormone and stress treatments on the expression of *PMEI* family members was analyzed in silico. [35]. The microarray data analysis of the *PMEI* family showed that several *PMEI* are differentially expressed in response to abscisic acid, gibberellic acid, auxin and methyl jasmonate treatment, and many are involved in cold, drought, heat, oxidation, salt, and wounding regulation in *Arabidopsis* [35]. In the same study, a *cis*-element analysis of promotor regions of the *Brassica campestris* ssp. *chinensis* *PMEI* gene family also indicated an involvement of several *PMEI* genes in environmental stress responses. For example, of the 100 *PMEI* genes in *Brassica campestris*, 38 *BcPMEI* promotors contained both abscisic acid and auxin (indole-3-acetic acid) related *cis*-elements, 63 and 35 promotors contained heat stress elements and low-temperature response elements, respectively. Additionally, MYB binding sites involved in drought stress were found in 58 *PMEI* promotors [35]. However, these in silico results will have to be validated by experimental data.

An *Arabidopsis* mutant with reduced expression of a *PMEI* gene (At1g62760) exhibited reduced sensitivity to salt stress [88]. Enhanced root growth, higher fresh weight and less chlorosis and necrosis upon NaCl treatment were accompanied with reduced salt stress signaling in the mutant, suggesting this PMEI as a negative regulator of salinity tolerance. In contrast, overexpression of *AtPMEI3* (At5g62360) in *Arabidopsis* also led to increased salt tolerance, as transgenic lines showed higher rates of germination, root growth, and survival under salinity conditions as compared to wild type plants [89]. This phenotype was also observed in plants overexpressing the closest homolog *CbPMEI1* from *Chorispora bungeana*, an alpine plant highly tolerant to chilling and freezing stress. Both genes were repressed by salt stress and abscisic acid but were induced by cold, suggesting distinct roles of the genes in freezing and salinity tolerance. The transgenic overexpression plants showed decreased freezing tolerance; however, they exhibited increased root growth under cold conditions. Plants increase pectin levels in the cell wall under low temperatures [90] probably to reduce cell wall porosity and increase cell adhesion. It was previously reported that DM of pectin increases during cold acclimation in pea [91]. In contrast, loss of AtPME41 in *Arabidopsis* by a transfer DNA insertion caused
an increase in freezing sensitivity [92]. Since pectins are important under low temperature conditions, PMEI activity could play a role in balancing the trade-off between freezing tolerance and growth by modifying the mechanical properties of cell walls.

**5. Role of PMEIs in Development**

PME-mediated changes in methyl-esterification of HG have been shown to affect various vegetative and reproductive stages of plant development (for reviews, see [6,7]). Therefore, we aim to give an overview of the latest results of molecular studies on PMEIs in plant development.

5.1. *PMEI Function in Seeds*

Upon hydration, mature *Arabidopsis* seeds rapidly extrude a gelatinous substance from seed coat epidermal (SCE) cells called mucilage, which is mainly composed of cell wall polysaccharides [93]. This layer has adhesive properties and possibly serves as a water-reservoir, which might facilitate seed germination, although it has been shown that mucilage production is not necessary for germination or plant fitness in the laboratory [94,95]. The mucilage is mainly composed of pectin, RG-I and small amounts of HG, but hemicellulose and cellulose have also been identified as minor components, as well as arabinogalactan proteins [96]. Several studies provided evidence that the DM of HG determines mucilage properties. For example, seven PME genes are specifically expressed in *Arabidopsis* seeds and the *pme58* mutant caused an altered pectin distribution in the mucilage [97]. Additionally, an E3 ubiquitin ligase, FLYING SAUCER1, was proposed to regulate the DM and a mutation lead to reduced mucilage extrusion and increased mucilage adherence [29]. Finally, the subtilase *atsbt1.7* mutant didn't show significant sugar compositional changes in its mucilage but a decrease in mucilage methylation, which was correlated with an increased PME activity [98].

More direct evidence was obtained by investigating the role of *PMEI6*, which was shown to be essential for proper mucilage release [44]. In three *pmei6* mutants, the outer primary cell wall of the SCE cells did not rupture into fragments but remained attached, which caused a delay in mucilage extrusion. This was accompanied by a lack of methylesterified HG (detected by antibodies) and reduced methanol release in the mucilage, as well as increased PME activity in seeds. The reduction in methylesterification in the outer primary cell wall of the three *pmei6* mutants could possibly lead to more abundant Ca²⁺ cross-linking of HG, which causes stronger attachment between adjoining cell walls and therefore impairs release of the mucilage. Further evidence indicates that PMEI14 is also involved in regulating pectin methyl-esterification in seed coat mucilage and that *PMEI14* together with *PMEI6* and *ATSBT1.7* are likely transcriptionally controlled by MYB52 [45]. Knock out mutants of *PMEI14* had an increased PME activity in seed mucilage but not in demucilated seeds. A reduced level of methylesterification of the mucilage was accompanied by an increased amount of calcium cross-linked HG detected by immuno-labelling. These studies show that PME16 function affects both seed coat mucilage and the primary SCE cell wall, and is necessary for proper mucilage release. In contrast, PMEI14 function is confined to inhibiting PME activity, specifically in the seed coat mucilage, potentially influencing pectin structure.

PME activity has also been shown to be tightly controlled during germination in *Arabidopsis* and garden cress (*Lepidium sativum*) [99,100], probably leading to cell wall weakening to allow radicle emergence. A small number of PME and PMEIs are strongly up-regulated upon germination, also in the presence of abscisic acid, which delays endosperm weakening and rupture [100]. Furthermore, overexpression of the PME inhibitor *PMEI5* resulted in a higher DM of seeds and reduced PME activity, which was accompanied by an earlier and faster germination process compared to wildtype [99]. This suggests that PMEI activity is essential for proper timing of seed germination but the precise molecular events are only beginning to be understood, based on modern techniques such as Chip-Seq [45] and potentially based on novel probes to dynamically monitor cell walls [101].
5.2. PMEI Function in Growth Processes

Plant cell growth is regulated by the interplay between the intracellular turgor pressure and the flexibility of the cell wall. Recent reviews have highlighted the importance of pectin, and especially HG, for mediating changes in the mechanical properties for control of growth processes [4,6,102]. In addition, studies using atomic force microscopy provided evidence that increased pectin de-methylesterification correlates with a decrease in cell wall stiffness [21,25].

A PMEI from *Arabidopsis*, AtPMEI4, has been shown to be involved in regulating hypocotyl growth. Growth acceleration of dark-grown hypocotyls requires significant cell wall remodeling, which is associated with transcriptional upregulation of pectin-modifying genes [103]. AtPMEI4 was upregulated during this developmental transition and its expression was shown to be specific for rapidly elongating epidermis cells in hypocotyls and roots. In an overexpression line, the onset of hypocotyl growth acceleration was delayed, suggesting that the PME inhibitor controls pectin de-methylesterification essential for the timing of the growth acceleration, but not the growth process itself [103]. On the other hand, *OsPMEI28* overexpression in rice had an effect on the growth process, namely inhibition of culm elongation and decreased cell wall thickness of culms, which resulted in a dwarfed phenotype [61]. Conversely, a *pmei4* mutant with elevated PME activity in *Arabidopsis* root cell walls showed an increase in root length [62], suggesting a correlation between increased de-methylesterification and elevated growth.

Controversial results have been reported for purified kiwi PMEI applied on *Arabidopsis* root tips, where an increase of root growth rate was stated upon inhibition of PME activity [104]. Similarly, *Arabidopsis* plants overexpressing *AtPMEI2* show enhanced growth and increased biomass due to enhanced cell expansion [105] as well as increased root length due to stronger cell elongation [26]. Increased root length and area was also observed in *Arabidopsis* plants expressing a PMEI from cotton [39]. Therefore, higher levels of DM of pectin seem also to be able to promote growth and cell expansion.

This correlation was also observed in pollen tube growth. Pollen tubes undergo a fast and unidirectional growth at the apex dependent on ion (e.g., calcium) dynamics and PMEs have been shown to play a role in this process [106,107]. A pollen-specific PME inhibitor, AtPMEI2, was shown to inhibit PME activity in *Arabidopsis* [52]. Although the pollen-specific pectin methylesterase *AtPPME1* was expressed along the whole pollen tube, the localization of AtPMEI2 was exclusively restricted to the apex, the site of elongation. This supports the model that elongation requires higher DM of the cell wall, which is caused by inhibition of PME activity, specifically at the tip. Conversely, along the pollen tube, PME activity is less inhibited, resulting in de-methylesterification, which in turn would lead to formation of calcium cross-links due to the availability of free calcium ions, causing increased cell wall stiffness. Corroborative, expression of *AtPME1* in tobacco pollen tubes inhibited elongation, whereas *AtPMEI2* expression led to an increase in elongation rate [52]. Antisense expression of a pollen-specific PMEI from broccoli (*Brassica oleracea*) in *Arabidopsis* triggered silencing of the orthologous *Arabidopsis* gene At1g10770 and resulted in male sterility. This suggests that disturbance of PME activity imbalances the pectin distribution and affects cell wall dynamics in the pollen cell wall with detrimental effect on fertility [108]. In addition, wheat *Tdpmei2.1* and *Tdpmei2.2* transcripts have been shown to undergo complete mRNA processing, specifically in anthers [42]. The occurrence of this mechanism suggests a role of the PME inhibitors during pollen development and reproductive processes, but requires further investigations.

The present data indicate a complex relationship between changes of DM of pectin (based on PMEI activity) and growth processes. Further studies are necessary to elucidate in a tissue-specific manner how the interplay of PMEs and PMEIs regulate pectin methylesterification and affect mechanical properties of cell wall necessary for cell elongation.
### 5.3. PMEI Function in Organ Formation

The mechanical properties of cell walls have been proposed to play a key role in plant morphogenesis and the control of organ outgrowth [7,109,110]. Organ morphogenesis requires a transition from isotropic growth of undifferentiated cells to anisotropic growth pattern, which is dependent on local cell wall loosening and stiffening by e.g., modification of cell wall components.

*AtPMEI3*, which is expressed in the apical meristem, was inducible expressed in *Arabidopsis*, which led to hyper-methylesterification of HG throughout the meristem dome upon induction [24]. As a result, the formation of both floral and flower organ primordia was inhibited. Using atomic force microscopy, it was shown that the reduced de-methylesterification of HG was accompanied by increased stiffness of cell walls in the shoot apical meristem in the *AtPMEI3* overexpressing lines [25]. This further resulted in shorter cells and loss of growth asymmetry, showing that anisotropic growth requires an asymmetric loosening of longitudinal walls, which is caused by fine-tuned de-methylesterification of pectin.

Constitutive overexpression of *AtPMEI5* in *Arabidopsis* also caused strong morphological phenotypes [111,112]. The stems of the transgenic plants showed an aberrant, twisted growth pattern, especially at branching points and branching organs (e.g., leaf or inflorescence), which failed to separate properly from the main stem [111]. Additionally, root growth was disturbed in the mutant and curled leaves and misshapen siliques were observed, which was proposed to be a result of activated BR signaling [112]. This indicates that PMEIs are involved in the fine-tuning of cell wall biomechanical properties via HG methylesterification that is required for proper organ formation and separation.

PMEIs might also play a role in organ senescence as changes in pectin methylesterification have been described, e.g., for petal senescence [113,114]. However, so far, only transcriptional changes of putative invertase/pectin methylesterase inhibitor genes in petals during senescence have been reported and further, more detailed studies will be necessary to investigate their putative function.

### 5.4. PMEIs in Fruit Development

Fruit ripening is a highly controlled developmental event during which the fruit undergoes various changes, e.g., in sugar contents, color, flavor, aroma, texture. Softening is a very (economically) important physiological change and several cell wall-modifying enzymes are involved in this process [115]. The first inhibitor of PME was identified in ripe kiwi fruit by Balisteri et al. [33] and was found active only in the mature stage, whereas it was undetectable in unripe fruits [116]. Furthermore, the expression levels of the *PMEI* genes in kiwi fruit increased with the progression of fruit maturation [117]. In banana (*Musa acuminata*), a PME inhibitor, *MaPMEI1*, was shown to be specifically expressed in the fruit and up-regulated during the ethylene-dependent ripening process [46]. Similarly, a PMEI has been identified in the tomato fruit [59]. *SolyPMEI* was strongly expressed in red fruits and, in lower levels, also in flowers and pollen, but not in green fruits. Interaction with the tomato PME-1 enzyme in the fruit and increased *SolyPMEI* expression levels with progression of ripening stages implicates the inhibitor in modulating PME activity and degree of methyl-esterification during fruit ripening in tomato. In grape (*Vitis vinifera*), *VvPMEI1* was found to be expressed only during the early phases of berry development, characterized by a rapid berry growth both through cell division and expansion [41]. *VvPMEI1* could be involved in regulating PME activity required for rapid cell growth and to maintain firmness, or it could prevent early softening of grape berries related to pectin degradation. This indicates a role for PMEIs in modulating PME activity and pectin methylesterification in different stages of fruit development.

## 6. Potential of PMEIs for Applications

Lignocellulosic biomass constitutes a sustainable renewable source of chemicals and fuels, but a major bottleneck for an industrial utilization is the natural recalcitrance of the cell wall due to its complex and heterogeneous structure. Modification of cell wall polysaccharides can improve the degradation into soluble sugars, which can be used by microorganisms for fermentation, a process
called saccharification [118]. Improved saccharification can make an industrial process more efficient and reduce cost for pre-treatments of the biomass. Lionetti et al. [105] showed that overexpression of *AtPMEI2* in *Arabidopsis* leads to a higher enzymatic saccharification efficiency, indicating a reduced cell wall recalcitrance likely due to the higher degree of HG methylesterification. The same was observed for wheat plants expressing the kiwi *PMEI*, suggesting that both cellulose and hemicellulose are better digestible during enzymatic hydrolysis in the transgenic plants. As a side effect, transgenic *Arabidopsis* plants had a higher biomass yield due to enhanced cell expansion. Further, a negative correlation between the level of de-methylesterified HG and cellulose degradability was found for 24 *Arabidopsis* accessions [119]. The reduction of egg-box structures or other crosslinks of methylesterified HG might be responsible for the accessibility of cellulose fibers for enzymatic digestion, which in turn results in increased saccharification efficiency. This indicates the potential of PMEI as a HG modifying enzyme to improve plant tissues for saccharification by targeted mutation or in wide-scale selection and breeding of plants for further utilization (e.g., as biofuels or for production of chemicals).

PMEIs have been shown to play a role in ripening processes (see above). Therefore, a potential application of this inhibitor would be in food (fruit and vegetable) processing. For food technology, endogenous PME activity is highly relevant because it determines the physiochemical and functional properties of pectin, thereby affecting the quality of plant-derived food products. PME activity in fruit juice causes undesired phase separation because de-methylesterified HG forms calcium crosslinks, and the resulting calcium pectate is insoluble and precipitates [120]. Addition of PMEI to orange juice was shown to control PME activity even over long storage, thereby avoiding phase separation. Application of PMEI would reduce required thermal treatments during juice processing and should result in better flavor and product quality [120]. For development or optimization of food-processing steps, it would be beneficial to determine PME activity in plant tissues. A PMEI-based molecular probe was shown to successfully label plant PMEs in various plant tissues and moreover, to differentiate between active and inactivated PME molecules [121]. This application of PMEIs would give important information about presence and localization of active PMEs after different food processing steps (e.g., thermal or high pressure treatment, microwaving). PMEIs have also been proposed for application in wine and marc production [41]. Pectin de-methylesterification releases high levels of methanol during different stages of grape processing, greatly affecting the composition of the final product [122,123]. Here, PMEIs could be used to reduce methanol formation in grape must and marc, as well as in products derived by fermentation and distillation [124]. Apart from applications in food industry, manipulation of PMEIs in planta could also be of interest. Overexpression of *PMEIs*, in banana fruits for example, would not only control over-softening but also likely extend banana shelf-life [46]. In conclusion, PMEIs could be good tools to control endogenous PME activity during food production processes, thereby positively influencing product quality.

As mentioned above, PMEIs play an important role in the response to biotic and environmental stresses. Therefore, the inhibitors could be potential targets for genetic engineering, aimed to improve plant fitness. In cold as well as salt stress conditions, PMEIs might be beneficial for balancing growth maintenance with stress tolerance and overexpression of *PMEIs* would be a promising way to improve salt tolerance of crops [89]. In addition, PMEI activity has been shown to be essential for resistance against fungal and bacterial pathogens [26,79,83]. Therefore, it could be useful as a potential molecular marker in plant breeding programs targeted to the selection of crop varieties with durable resistance.

**Author Contributions:** Conceptualization, A.W.; Writing—Original Draft Preparation, A.W.; Writing—Review & Editing, A.W. & B.U.; Visualization, A.W.; Funding Acquisition, B.U. & A.W.

**Funding:** This research was supported by DFG (grant number: US98/13-1), the German Federal Ministry of Education and Research (grant number: 031B0193B) as well as the Ministry of Innovation, Science and Research of North-Rhine Westphalia within the framework of the North-Rhine Westphalia Strategieprojekt BioEconomy Science Center (grant number 313/323-400-00213).

**Conflicts of Interest:** The authors declare no conflict of interest
Abbreviations

<table><tr><td>PMEI</td><td>Pectin methylesterase inhibitor</td></tr><tr><td>PME</td><td>Pectin methyl esterase</td></tr><tr><td>DM</td><td>Degree of methylation</td></tr><tr><td>HG</td><td>Homogalacturonan</td></tr><tr><td>GalA</td><td>D-galacturonic acid</td></tr><tr><td>GAUT</td><td>Galacturonosyltransferases</td></tr><tr><td>OGs</td><td>Oligogalacturonides</td></tr><tr><td>DAMP</td><td>Damage associated molecular pattern</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub></td><td>Hydrogen peroxide</td></tr><tr><td>SCE</td><td>Seed coat epidermis</td></tr><tr><td>MUM1</td><td>Mucilage-modified mutant 1</td></tr></table>

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Review

# Overview of the Role of Cell Wall DUF642 Proteins in Plant Development

José Erik Cruz-Valderrama<fnref n="1" />, Ximena Gómez-Maqueo<fnref n="1" />, Alexis Salazar-Iribe<fnref n="1" />, Esther Zúñiga-Sánchez<fnref n="1" />, Alejandra Hernández-Barrera<fnref n="1" />, Elsa Quezada-Rodríguez<fnref n="1" /> and Alicia Gamboa-deBuen<fnref n="2" />,*

Received: 30 May 2019; Accepted: 4 July 2019; Published: 6 July 2019

**Abstract:** The DUF642 protein family is found exclusively in spermatophytes and is represented by 10 genes in Arabidopsis and in most of the 24 plant species analyzed to date. Even though the primary structure of DUF642 proteins is highly conserved in different spermatophyte species, studies of their expression patterns in Arabidopsis have shown that the spatial-temporal expression pattern for each gene is specific and consistent with the phenotypes of the mutant plants studied so far. Additionally, the regulation of DUF642 gene expression by hormones and environmental stimuli was specific for each gene, showing both up- and down-regulation depending of the analyzed tissue and the intensity or duration of the stimuli. These expression patterns suggest that the DUF642 genes are involved throughout the development and growth of plants. In general, changes in the expression patterns of DUF642 genes can be related to changes in pectin methyl esterase activity and/or to changes in the degree of methyl-esterified homogalacturonans during plant development in different cell types. Thus, the regulation of pectin methyl esterases mediated by DUF642 genes could contribute to the regulation of the cell wall properties during plant growth.

**Keywords:** DUF642 family; homogalacturonans; plant cell wall; plant development; pectins

## 1. Introduction

Plant cell walls are dynamic compartments whose composition and chemical structure vary during plant development in response to environmental signals. Cell walls are composed of three layers: the middle lamella, and the primary and secondary cell walls. Primary cell wall deposition continues through cell growth and expansion. It is a very complex and dynamic structure composed mainly of three types of polysaccharides: cellulose, hemicelluloses, and pectins. Cellulose microfibrils and hemicelluloses constitute a network with structural proteins that are embedded in a gel-like matrix of pectins. Localization of different classes of polysaccharides within the cell wall appears to depend on species, organ, tissue, and cell type, and the pectin network is temporally and spatially regulated during plant development. Alterations of the cell wall structure and changes in properties during plant development are mainly the result of protein activities. Enzymatic activity and protein interactions respond to developmental and environmental signals and tightly regulate the complex and dynamic structure of plant cell walls.

In terrestrial plants, the chemical structure of homogalacturonans (HGs), a type of pectin that is comprised of chains of α-1,4-galacturonic acid, is modified by de-methyl-esterification processes during plant development by pectin methyl esterases, and pectin methyl esterase inhibitory proteins regulate the degree of HGs' esterification of the cell wall in different cell types. Consequently, cell or tissue-specific function could be influenced by the HGs' status [1]. An increase in complexity in

<footnote n="1">¹ Instituto de Ecología, Universidad Nacional Autónoma de México. Mexico City 04510, Mexico</footnote>

<footnote n="2">² Universidad Nacional Autónoma de México, Mexico City 04510, Mexico</footnote>

<footnote>* Correspondence: agamboa@ecologia.unam.mx</footnote>
the regulation of HGs' esterification status through plant evolution has been described including a diversification of the gene families involved either in pectin synthesis or in pectin modification [2,3]. The shift from an aquatic to a terrestrial environment constitutes a milestone in the complexity of pectin regulation. In the algae *Chara*, cell growth is a result of a calcium pectate cycle that does not involve enzymatic activities [4]. Charophytes are the closest related group to land plants, thus, this enzyme-less mechanism could be still present in terrestrial plants [5].

The DUF642 protein family is found exclusively in spermatophytes and is involved in regulating HGs esterification. The DUF642 family was first described in apoplast proteomes of rosette leaves in *Arabidopsis thaliana* (Arabidopsis) by Boudart et al. [6] and subsequently in cell wall proteomes from different tissues and plant species from both dicotyledons and monocotyledons [7,8]. This multigene family is represented by 10 genes in Arabidopsis as well as in other plant species [9]. DUF642 proteins contain two DUF642 domains (PF04642) that present two conserved motifs NGXFEXXP and CGPVXD, determined as the family signature. A phylogenetic analysis revealed four clades of orthologous genes for all spermatophytes (Clades A, B, C, and D). After the divergence of gymnosperms and angiosperms, a duplication of Clade A occurred, producing two subclades (A1 and A2), and a subsequent duplication occurred after the divergence of monocotyledons and eudicotyledons [9].

The DUF642 proteins are encoded by genes that contain three exons. The first exon encodes a signal peptide, and the other two exons encode the DUF642 domains. Proteomic and cellular localization studies of the Clade A proteins indicated they were located primarily in the cell wall [6,10]. However, the protein encoded by *At3g08030* was located in the nuclear pore, probably because this gene has an alternative first exon edition so one of the possible encoded protein lacks the signal peptide. The proteins in Clades B, C, and D have a GPI-anchor site at the carboxyl terminus and were detected in cell membrane proteomes [11].

The interactions of the DUF642 proteins with polysaccharides and cell wall enzymes have been demonstrated in vitro. The protein encoded by *At3g08030* (Clade A2) interacted specifically with cellulose [11]. The proteins encoded by *At4g32460/BDX* and *At5g11420* (Clade A1) interacted with the catalytic domain of a pectin methyl esterase (PME) [12]. In two transgenic Arabidopsis lines overexpressing *BDX* the PME activity increased in the seeds, seedlings, and the apical meristems, but no changes in PME activity were detected in the leaves [13]. PME activity was higher in the seeds of both overexpressing lines than it was in the control seeds during germination [13,14]. PME activity was also higher in seedlings, stems, leaves, and fruits in Arabidopsis plants overexpressing the *VqDUF642* gene (Clade A1) of *Vitis quinquangularis* [15]. The leaves of Arabidopsis plants overexpressing the *AhDGR2* gene (Clade A1) of *Amaranthus hypochondriacus* showed a decrease in PME activity, whereas an increase in PME activity was detected in the roots [16]. The phenotypes of all the Clade A1 protein mutants in Arabidopsis have been established. The *At5g25460/dgr2* mutant had a shorter root and smaller rosette leaves than the wild-type [17]. No alterations in the phenotypes of the *At5g11420* and *At1g80240/dgr1* mutants compared with the wild-type have been described. The *At4g32460/bdx* mutants had shorter siliques, seeds with altered development, and longer hypocotyl than the wild-type [18]. Among Arabidopsis Clade A2 mutants, the *At2g41800/teb* mutant is the only one for which a phenotype (longer hypocotyls) has been established [19]. No phenotypes have been established for the two Clade B genes *At1g29980* and *At2g34510*, or the Clade D gene *At5g14150*.

## 2. Expression Patterns of DUF642 Genes

The expression patterns of the Clade A *DUF642* genes have been studied in Arabidopsis plants transformed with constructs containing the putative promoter region fused to the reporter genes *ER-GFP* and *GUS* (Figure 1). Differential expression patterns were detected at different stages of plant development (Table 1).
<figure><img src="image_114.png" /><figcaption>**Figure 1.** Expression patterns of the *DUF642* genes described to date using reporter genes during Arabidopsis plant development.</figcaption></figure>

In Arabidopsis during embryonic development, *BDX* and *At3g08030* were expressed from the heart stage onwards. In the late heart stage, *At3g08030* was expressed in the quiescent center and in the columella cells, while *BDX* was expressed in the vascular tissue (Figure S1) [13].

During seedling development in Arabidopsis, *BDX*, *At5g11420*, *DGR2*, *TEB*, and *At3g08030* were expressed in hypocotyls and *BDX* and *DGR2* were also expressed in cotyledons, but no expression was detected for *At5g11420*, *TEB*, and *At3g08030* [13,17,20]. In hypocotyls, specific expression of *BDX*, *At5g11420*, *At3g08030*, and *TEB* in the epidermal cells was determined by confocal microscopy. *DGR1* and *At2g34510* expression was detected specifically in hypocotyls grown under shade conditions [21]. Transcriptomic studies in *Brassica rapa* indicated that three orthologous genes of *BDX*, *At5g11420*, and *DGR2* were expressed in hypocotyls [20].

The expression patterns of only *DGR1*, *BDX*, and *DGR2* have been studied in leaves of Arabidopsis. *BDX* expression was localized in the vascular tissue, *DGR2* expression was located in the leaf primordium, and *DGR1* expression was not detected in the leaves [13,17].

In flowers of Arabidopsis, *BDX* was expressed in petals, vascular tissue of the filament, anther, and stigmatic papilla. *At5g11420* was expressed in petals and abscission zones of floral whorls, *TEB* was expressed only in stigmatic papilla, and *At3g08030* was expressed in the replum of androecium and epidermis of the filaments of stamens [13,18] (Figure S1).

In Arabidopsis, *DGR1*, *BDX*, *At5g11420*, *DGR2*, *TEB*, and *At3g08030* expression was detected in the primary root. *BDX*, *At5g11420*, *DGR2*, *TEB*, and *At3g08030* were expressed in epidermal cells of the meristematic region. *BDX* also was expressed in vascular tissue and *At3g08030* was expressed in the quiescent center and in columella [13,17,18] (Figure S1). These expression patterns were similar to those found during embryonic development.

During development of the lateral roots, *BDX* and *At3g08030* expression was detected from stage II onwards, when cell proliferation begins (Figure 2). Further, at the moment of radicle protrusion, *At5g11420* and *TEB* expression was detected in the epidermal cells that surround the radicle protrusion zone. The expression patterns of *DGR1* and *DGR2* during this process have not been determined.

The expression patterns of the Clade A *DUF642* genes were altered by plant hormones (Table 1 and Table S1). For example, auxins and gibberellins increased the expression levels of *BDX* and *TEB* during Arabidopsis seed germination [22]. Exogenous auxins and gibberellins also altered the expression pattern of *BDX* by promoting its expression in cortex cells in addition to vascular tissue of Arabidopsis. The expression pattern of *At3g08030* was not altered by the addition of auxins [13].
<table><caption>**Table 1.** Expression profiles of the *DUF642* genes detected in distinct tissues/organs and species. *DUF642* genes are grouped by clades as reported by Vázquez-Lobo et al. (2012) [9] and using the locus tag of Arabidopsis as a reference for gene grouping.</caption><thead><tr><th>Clade</th><th>Gene Name</th><th>Species</th><th>Expressed in Tissue/Organ</th><th>Reference</th></tr></thead><tbody><tr><td>A1</td><td>At1g80240</td><td>Arabidopsis</td><td>Petal</td><td>[23]</td></tr><tr><td>A1</td><td>GRMZM2G027683</td><td>Zea mays</td><td>Embryo</td><td>[24]</td></tr><tr><td>A1</td><td>OSNPB_040494600</td><td>Oryza sativa</td><td>Floral buds, flowers, mature seed, milk grains, roots before flowering</td><td>[25]</td></tr><tr><td>A1</td><td>Orthologous to At4g32460</td><td>Brassica rapa</td><td>Hypocotyl epidermal cells</td><td>[21]</td></tr><tr><td>A1</td><td>Orthologous to At4g32460</td><td>Cannabis sativa</td><td>Stem (apical section)</td><td>[26]</td></tr><tr><td>A1</td><td>OSNPB_020205200</td><td>Oryza sativa</td><td>Floral buds, flowers, mature seed, milk grains</td><td>[25]</td></tr><tr><td>A1</td><td>At5g11420</td><td>Brassica rapa</td><td>Hypocotyl epidermal cells</td><td>[21]</td></tr><tr><td>A1</td><td>At5g11420</td><td>Arabidopsis</td><td>Petal</td><td>[23]</td></tr><tr><td>A1</td><td>At5g11420</td><td>Arabidopsis</td><td>Root (cortex)</td><td>[27]</td></tr><tr><td>A1</td><td>OSNPB_010611000</td><td>Oryza sativa</td><td>Floral buds, flowers, mature seed, milk grains, roots before flowering</td><td>[25]</td></tr><tr><td>A1</td><td>OSNPB_040494800</td><td>Oryza sativa</td><td>Floral buds, milk grains, roots before flowering</td><td>[25]</td></tr><tr><td>A1</td><td>Orthologous to At5g25460</td><td>Brassica rapa<br>Amaranthus<br>hypochondriacus</td><td>Hypocotyl epidermal cells</td><td>[21]</td></tr><tr><td>A1</td><td>AhDGR2</td><td>var. Revancha</td><td>Panicle, axillary bud, young stem, and young leaf</td><td>[16]</td></tr><tr><td>A1</td><td>GRMZM2G051571</td><td>Zea mays</td><td>Seed</td><td>[24]</td></tr><tr><td>A1</td><td>GRMZM2G034985</td><td>Zea mays</td><td>Seed, shoot, root, SAM, ear, tassel, cob, silk, anthers, ovule</td><td>[24]</td></tr><tr><td>A1</td><td>Orthologous to At5g25460</td><td>Cannabis sativa</td><td>Stem (apical section)</td><td>[26]</td></tr><tr><td>A1</td><td>At5g25460</td><td>Arabidopsis</td><td>Root (cortex)</td><td>[27]</td></tr><tr><td>A2</td><td>At3g08030</td><td>Brassica rapa</td><td>Hypocotyl inner tissue cells</td><td>[21]</td></tr><tr><td>A2</td><td>At3g08030</td><td>Arabidopsis</td><td>Root (cortex/hair cell)</td><td>[27]</td></tr><tr><td>A2</td><td>At2g41800</td><td>Arabidopsis</td><td>Carpel</td><td>[23]</td></tr><tr><td>A2</td><td>OSNPB_030807700</td><td>Oryza sativa</td><td>Floral buds, flowers, mature seed, milk grains, roots before flowering</td><td>[25]</td></tr><tr><td>A2</td><td>At2g41810</td><td>Brassica rapa</td><td>Hypocotyl epidermal cells</td><td>[21]</td></tr><tr><td>B</td><td>Orthologous to At1g29980</td><td>Brassica rapa</td><td>Hypocotyl inner tissue cells</td><td>[21]</td></tr><tr><td>B</td><td>OSNPB_010756600</td><td>Oryza sativa</td><td>Floral buds, flowers, leaves before flowering, mature seed, milk grains, roots before flowering</td><td>[25]</td></tr><tr><td>B</td><td>GRMZM2G324705</td><td>Zea mays</td><td>Seed, shoot, root, SAM, ear, tassel, cob, silk, anthers, ovule</td><td>[24]</td></tr></tbody></table>
<figure><img src="image_115.png" /><figcaption>**Figure 2.** Expression patterns of *At3g08030* during lateral root development. The Arabidopsis *pAt3g08030::ER-GFP*-transgenic plants were obtained as described in Salazar-Iribe et al. [19]. Roman numerals indicate the developmental stages of the lateral root initiation process. The images are all section images. n = 8–10 roots for each developmental stage. Scale bars = 20 µm.</figcaption></figure>

**3. Subcellular Localization of DUF642 Proteins**

DUF642 proteins are very abundant in cell wall proteomes from different tissues and plant species [7,8]. The At3g08030 protein has been found in all Arabidopsis cell wall proteomes reported so far. TEB and DGR1 were found in the proteomes of cell suspension cultures, whereas DGR2 was found in the proteomes of cell suspension cultures, hypocotyls, and mature stems. The At5g11420 protein was present in the proteomes of apoplast, hypocotyls, and mature stems, whereas BDX was found only in the mature stem cell wall proteome [28]. The localization of BDX and TEB in the Arabidopsis cell wall was confirmed by confocal microscopy in hypocotyl cells [18,19]. The VqDUF642 protein was localized in the cell wall of tobacco epidermal cells [15] and peach PpDU642 (TEB) was localized in the cell wall and extracellular space during a transient expression in tomato fruit [29].

Studies carried out to determine the subcellular localization of BDX in epidermal root cells of Arabidopsis suggested that it was localized intracellularly, probably in Golgi, and then relocated to the cell wall when the plants were subjected to abiotic (NaCl) or biotic (nematodes) stress stimuli [30] (Figure S2). TEB is highly expressed during cell division prior to cytokinesis. In Arabidopsis, the synchronization of mitosis in primary root cells treated with hydroxyurea [31] confirmed that TEB was localized intracellularly during mitosis, and was located in the cell wall until the end of the process (Figure 3). These results suggest that BDX and TEB are located transiently in the cell wall in response to endogenous or exogenous stimuli. Finally, polar localization of TEB in the cell wall of radicular hair was determined during its elongation, suggesting the formation of domains with an accumulation of these proteins within the cell walls [19].
<figure><img src="image_116.png" /><figcaption>**Figure 3.** Subcellular localization of the TEB protein in meristematic root cells synchronized with hydroxyurea. (A) Arabidopsis *pTEB::TEB-GFP* seedlings were treated with hydroxyurea for 16 h, and a diffuse pattern of GFP in meristematic cells of the cortex was observed. The cell wall was stained with propidium iodide (red). (B) After 18 h of hydroxyurea treatment, GFP was detected as a ring surrounding the nucleus in the cortex cells. (C) TEB was located in the cell wall after 19 h of hydroxyurea treatment. Hydroxyurea stops DNA synthesis and after 16–17 h most of the cells are at the G2/M transition and at 18–20 h cytokinesis occurs according to Cools et al. [31]. Seedlings were sown in MS medium and transferred on day 6 to MS medium with hydroxyurea for 16–20 h. The images are all section images. Scale bars = 20 µm. The Arabidopsis transgenic plants were obtained as described in Salazar-Iribe et al. [19].</figcaption></figure>

# 4. Function of DUF642 Proteins in Plant Development

The expression patterns of *DUF642* genes suggest they are involved throughout the development and growth of plants. In general, changes in the expression patterns of *DUF642* genes can be related directly to PME activity [13,15,16,19]. The leaves of Arabidopsis lines overexpressing *AHDGR2* had low PME activity, and this was reflected in alterations in the pattern of de-methyl-esterified HGs in the cell walls [16,19,32].

## 4.1. *Seed Development*

In Arabidopsis, *BDX*, *At5g11420*, *TEB*, and *At3g08030* were expressed during embryo development (Table 1 and Table S1), and the localized expression during development indicated differential expression in different cell types (Figure 1). However, the *bdx-1*, *At2g11420*, and *teb-1* mutants did not show alterations in gene expression during embryo development.

During endosperm development, *BDX* was the only *DUF642* gene reported at four days after pollination [33]. Of the *dgr1*, *bdx-1*, *At5g11420*, *dgr2*, and *teb-1* mutants studied during seed development, only the *bdx-1* mutant showed alterations, producing misshapen seeds with changes in the folding of the embryo. The endosperm and seed coat exert physical restrictions during elongation and folding of the embryo [34], with the walls of the endosperm adjacent to the embryo showing a decrease in the degree of HGs esterification. In the *bdx-1* mutant, a lower signal of de-methyl-esterified HGs compared with the wild-type was detected in this region. Alterations in the degree of esterification can cause greater rigidity of the cell wall of endosperm cells, thereby generating a physical restriction in the development of the embryo and compromising the subsequent survival of the seedling [32].

The accumulation of *DUF642* gene transcripts has also been reported in mature seeds. Germination pre-treatments such as priming, which improve germination performance, can induce the expression of these genes. An important increase in the expression of the orthologous gene of *At3g08030* was reported in seeds from *Brassica oleracea* subjected to an osmopriming treatment [35]. In Arabidopsis, *Ceiba aesculifolia*, and *Wigandia urens* seeds, the expression of *At3g08030* increased in response to osmopriming treatment and decreased when the seeds were subjected to controlled deterioration [36]. In a study carried out with Arabidopsis *vps29* mutant seeds (VPS29 rearrangement complex), which had a lower germination index, the abundance of *At3g08030* and *At1g29980* increased while that of *At5g11420* and *DGR2* decreased [37]. Functional studies of *At3g08030* loss of function mutants could reveal if this gene has a role in promoting seed longevity.
After seed maturation, the expression of *At5g11420*, *DGR2*, *At3g08030*, and *At1g29980* increased in after-ripened seeds (Table S1). These changes in expression may be related to changes in the biochemical machinery that are required to modify the architecture of the cell wall of the embryo, the endosperm, and the seed coat during germination.

## 4.2. Germination

During the germination process of barley, Brassica, and Arabidopsis seeds, progressive differential expression of several *DUF642* genes took place (Table S1). The expression levels of Clade A1 genes (*BDX*, *At5g11420* and *DGR2*) and *At3g14310*, which codes for PME3, increased after 6 h of seed imbibition. The expression of the Clade A2 gene, *TEB*, increased between 8 and 12 h, and *At3g08030* expression was detected in the mature seeds and during the entire imbibition process (Figure S3).

The lines overexpressing *BDX*, *At5g11420*, and *DGR2* showed a decrease in the onset of germination, especially in the timing of the testa rupture, compared with the wild-type seeds [13] (Figure S3). The increase in total PME activity during seed germination in the overexpressing plants may be related to the early testa rupture described previously [14]. Early testa rupture also was observed in tomato seeds from tomato plants overexpressing *VqDUF642* [15]. These results indicate that the Clade A1 genes have the same function during germination.

## 4.3. Hypocotyls

Under control growth conditions (long photoperiod), the Arabidopsis *bdx-1* and *teb-1* mutant lines had longer hypocotyls because of longer cell length compared with the wild-type, whereas the overexpressing lines produced shorter hypocotyls. The hypocotyls of the *bdx-1* and *teb-1* mutants had a higher signal for methyl-esterified HGs. Additionally, the *bdx-1* mutant accumulated more auxins in the epidermal cells than the wild-type [18]. During cell elongation in the hypocotyl, auxin signaling and cell wall modifications of epidermal cells were actively involved [38,39]. The accumulation of auxins in hypocotyl epidermal cells during cell elongation was facilitated by PIN transporters. In the *bdx-1* mutant, a change in the localization of PIN1 transporters was detected, possibly because of the degree of HGs esterification, as has been described for other tissues [40]. The *bdx* and *teb* mutants showed an altered hypocotyl phenotype, suggesting *DUF642* genes may be involved in regulating hypocotyl elongation during seedling development [41].

## 4.4. Leaves

The expression of *DGR1*, *BDX*, *At5g11420*, *DGR2*, *TEB*, *At2g41810*, *At3g08030*, *At2g34510*, and *At5g14150* was detected in rosette leaves of mature Arabidopsis plants close to flowering (Table S1). The *dgr1*, *bdx-1*, and *teb-1* mutants showed no alterations in the development of rosette leaves. The *dgr2* mutant had a smaller rosette, explained by its expression in leaf primordia [17]. The lines overexpressing *BDX*, *At5g11420*, and *TEB* showed no visible alterations in the leaves or in the size of the rosette [13,19]. No differences were found in the leaves of Arabidopsis plants overexpressing *At1DGR2* or tomato plants overexpressing *VqDUF642* [15,16]. The mechanisms that control leaf origin and growth are complex, because of the phenotypic plasticity that is present in leaves as a mechanism for adaptation to different environments [42]. The complexity of the regulation of leaf size and morphology, and the lack of alterations in the leaves of mutant lines suggest the *DUF642* genes are functionally redundant in leaves.

## 4.5. Reproductive Structures

During flower development in Arabidopsis, the *DUF642* genes studied to date showed differential expression patterns, although the expression of only two genes have been detected in carpel, stamens, and petals. *BDX* is the only *DUF642* gene that has been detected in the anthers of stamens. The *bdx-1* mutant line showed morphological alterations of the pollen grain that altered its viability. The in vitro germination of the pollen tube was lower in the *bdx-1* mutant pollen grains that in the wild-type.
The structural and functional alterations of the pollen grains in the *bdx-1* mutant could be caused by changes in the accumulation of auxins during the late stages of anther development (Figure 4). Similar alterations in pollen morphology have been described in mutants related to the auxin signaling pathway, including auxin synthesis and transport [43].

<figure><img src="image_117.png" /><figcaption>**Figure 4.** Pollen grain development and auxin accumulation in anthers of flowers from wild-type and *bdx-1* mutant Arabidopsis plants. Normal pollen grain development in wild-type (wt) flowers. (A) Anthers from 7–8 stage flower. (B) Anthers from 11–12 stage flower. (C) Anthers from 13 stage flower. GFP detection of auxins in anthers from wt/DR5 flowers. (D) Anthers from 7–8 stage flower. (E) Anthers from 11–12 stage flower. (F) Anthers from 13 stage flower. Throughout the development of the anther there is an important accumulation of auxins. Pollen grain development in bdx-1/DR5 flowers. (G) Anthers from 7–8 stage flower. (H) Anthers from 11–12 stage flower. (I) Anthers from 13 stage flower. The development of pollen grains is altered; at the 11–12 stage, diffuse cytoplasm is observed and, at stage 13, some pollen grains have collapsed. GFP detection of auxins in anthers from bdx-1/DR5 flowers. (J) Anthers from 7–8 stage flower. (K) Anthers from 11–12 stage flower. (L) Fertilized flowers at stage 13. Throughout the development of the anther there is almost no accumulation of auxins. The Arabidopsis transgenic plants were obtained as described in Cruz-Valderrama et al. [32]. Sections: 1–2 µm. Staining was with toluidine blue. Red bar = 20 µm, Black bar = 10 µm. n = 8–10 plants for each line. White bar = 100 µm. n = 5–9 anthers from flowers from different plants.</figcaption></figure>

### 4.6. Fruits

Studies of *DUF642* gene expression and gene function during fruit development are very scarce. Of the Arabidopsis mutants studied, only *bdx-1* showed a fruit phenotype with shorter siliques and
lower seed production than wild-type plants. In the genus *Brassica*, *DGR2* is the only gene that has been detected in transcriptomes of siliques, specifically in the shells of the pods [44].

### 4.7. *Roots*

The expression of *DGR1*, *BDX*, *At5g11420*, *DGR2*, and *TEB* has been detected in primary roots of Arabidopsis. All these genes were expressed in the epidermal cells of the meristematic zone, except *DGR2*, which was expressed in the elongation zone. The *dgr2* mutant was the only one that presented a short root phenotype [17]. Overexpression of *AhDGR2* in Arabidopsis produced longer roots [16], which is understandable given that *DGR2* was expressed in the meristematic zone where the cells begin to expand and the rate of cell division is reduced.

The *bdx-1*, *At5g11420*, and *teb-1* mutants showed no alterations in the development of lateral roots. *BDX*, *At3g08030*, *At5g11420*, and *TEB* all had similar expression patterns throughout the development of lateral roots, which suggested there could be a functional redundancy that would explain the lack of altered phenotypes for these mutants.

Roots interact with microorganisms in the soil, and are particularly susceptible to pathogen attacks. Roots also are central in the perception of nutrient concentrations and other elements like heavy metals in the substrate where plants develop. The modification and restructuring of the cell wall in roots actively participate in the response to different types of stresses. Therefore, the participation of *DUF642* genes in the remodeling of the cell wall can be relevant in all these processes. Changes in the expression of *DUF642* genes in response to biotic and abiotic stresses have been described in various plant species (Table S2). However, very few functional studies are available related to the particular role of *DUF642* genes in response to stress.

## 5. Biotic Factors

*DGR2* is negatively regulated in Arabidopsis plants during infection by the bacterium *Ralstonia solanacerum* [45]. The expression of *At1g29980* and *At3g08030* is induced by infection with *Rhodococcus fascians* [46]. *At3g08030* also is highly expressed in response to infection by *Agrobacterium tumefaciens* [47].

The abundance of the DUF642 protein encoded by *At3g08030*, which has two isoforms with different isoelectric points, was reduced significantly in the cell wall proteome of Arabidopsis suspension cells treated with chitosan, an elicitor that mimics a fungal infection [48]. Infection with the *Penicillium* strain *Pc4* in post-harvest grapes increased the abundance of two isoforms of the *At5g11420* orthologous protein, probably because of post-translational modification [49]. In apoplastic proteomes of maize roots (*Zea mays*) infected with the symbiont species *Trichoderma virens*, a DUF642 protein was detected 5 days after inoculation. Modification and degradation of root cell walls are essential for colonization to take place [50]. Infection of Arabidopsis plants with the phytopathogenic fungus *Botrytis cinerea* reduced the expression of *At5g11420* and *At5g25460* [51].

The overexpression of *VqDUF642* in tomato plants reduced the susceptibility to *B. cinerea* infection in both mature and immature fruits. Overexpression of this gene modified the expression of some pathogen response genes such as *SIPR1*, *SIPR2*, *SIPR3*, and *SIPR4* whose expression increased at 48 h post-infection. In transgenic Thompson grape plants, overexpression of *VqDUF642* promoted increased resistance to *B. cinerea* and induced resistance to the fungus *Erysiphe necator*, which causes oidium of vines. The leaves of the overexpressing plants infected with *E. necator* showed a less severe infection than the wild-type at 48 h post-infection. In addition, the expression levels of the pathogen-response genes *VvPR1*, *VvPR2*, *VvPR3*, and *VvPR4* increased drastically at 48- and 96-h post-infection in transgenic seedlings compared with the wild-type [15].

In Arabidopsis, transcriptomic induction during early inoculation with the knot-nematode *Meloidogyne incognita* revealed up-regulation of *At1g29980* [52]. *BDX* and *TEB* expression were also highly induced by *M. incognita* early inoculation. The cell wall localization of *BDX* and *TEB* in the epidermal cells of primary roots was induced by *M. incognita*. Early inoculation with *Nacobus aberrans*, a nematode that cannot infect Arabidopsis, did not alter the expression of these two genes [30].
A comparative analysis of available Arabidopsis flower transcriptomes showed that changes in the expression patterns of flower-specific defense genes were critical in pathogen resistance. According to this study, the expression of *BDX* and *DGR2* was positively regulated in petals compared with in senescent leaves, in stage 15 of the flower. At this stage, an increase in the expression of the biotic stress response genes also occurred [53].

## 6. Abiotic Factors

The regulation of the DUF642 genes by transcription factors in response to aluminum stress has been reported. Aluminum stress has been studied extensively in aluminum-resistant plants. In particular, *Oryza sativa* (rice) is a cultivated crop plant that is resistant to aluminum, and the signaling pathway associated with its resistance has been studied widely. The Al resistance transcription factor 1 gene (*ART1*) is central to the aluminum response, and it regulates an increase in the expression of the DUF642 gene *Os04g049490* (orthologous to *At5g11420*) [54]. *Os04g049490* was also regulated by the *SENSITIVE TO ALUMINUM RHIZOTOXICITY* gene (*STAR1*), which plays a fundamental role in aluminum resistance in rice roots [55]. In the *O. sativa indica* IR64 cultivar treated with aluminum, *Os04g41750* (orthologous to *At5g11420*) expression was up-regulated, whereas no change in its expression was detected in *O. sativa* cv. Azucena, which is more sensitive to aluminum [56]. Plants in the genus *Stylosanthes* have high tolerance to toxicity by the aluminum ion, and expression of the orthologous DUF642 gene *Os04g0494900* increased in the roots of the Reyan 2 genotype in response to aluminum treatment [57]. A comparison between the transcriptomes of two *Citrus* species with different aluminum tolerances suggested that the process of HGs de-esterification in the cell wall of the root cells played an important role in resistance to aluminum. In *C. sinensis*, which is resistant to aluminum, four genes related to this process and a DUF642 gene, an ortholog of *DGR2*, were up-regulated [58]. In NtSTOP-1-RNAi tobacco plants that showed a decrease in the expression of the *SENSITIVE TO PROTON RHIZOTOXICITY* gene (*STOP1*), the expression of *Nt6860* (orthologous to *At5g11420*) decreased, as has been described for *At5g11420* in the Arabidopsis *art-1* mutant [59].

In *Medicago sativa* plants exposed to cadmium, the amount of the protein orthologous to the Arabidopsis protein, DGR2, decreased [60]. The exposure of *Populus × canadensis* plants to high doses of zinc promoted the decrease in the expression of *POPTR_0001s27110* (orthologous to *At3g08030*) [61].

Ultraviolet radiation (UV-B) generates an imbalance in the production of reactive oxygen species. Thus, it is used to study the effects of oxidative stress on plants. In the mutant *ggt1* of the protein GAMMA-GLUTAMYL TRANSFERASE ISOFORM 1(GGT1), involved in the redox balance, the proteins DGR2 and PECTIN METHYL ESTERASE3 (At3g14310) are very abundant in both the mutant and in the wt of Arabidopsis plants exposed to UV-B [62].

In the salinity-resistant plant *Manihot esculenta* Crantz (Cassava), *RknMes02_00171* and *RknMes02_00443*, which are orthologs of *BDX* and *At5g11420* respectively, were among the 40 most highly expressed genes during NaCl treatment [63]. *AhDGR2* was significantly induced in *Amaranthus hypochondriacus* (grain amaranth), which is also resistant to high doses of salt. However, when *AhDGR2* was overexpressed in Arabidopsis, the plants showed hypersensitivity to increasing NaCl concentrations, as shown by shorter root length, smaller and slightly chlorotic rosettes, as well as considerably reduced germination rates [16].

The expression levels of *At2g34510* and *At5g11420* increased in Arabidopsis plants subjected to drought stress, although their expression levels were not altered by treatment with abscisic acid [64]. The increase in *At2g34510* expression induced by drought was corroborated in a study that tested the stress response in natural populations of Arabidopsis [65]. In *Eucalyptus calmadulensis*, the expression of *Eucgr.C02812* (orthologous to *At5g11420*) was also found to decrease in plants exposed to drought [66].

## 7. Conclusions

The DUF642 proteins are highly conserved, which may be related to their specific interactions with cell wall polysaccharides and proteins in different cell types. The *At3g08030*-encoded protein
interacts in vitro with cellulose, a polysaccharide that has an important role in plant development [9]. However, there are still no functional studies that confirm this interaction. BDX and At5g11420 interact in vitro with a PME [12]. Studies in different plant species suggest that their function could be related to the regulation of HGs modification throughout plant development. The functional studies carried out to date are consistent with this hypothesis. For instance, in almost all plant tissues where *DUF642* gene overexpression has been induced, there was an increase in the total PME activity. Furthermore, the increase in methyl-esterified HGs in the endosperm during embryo folding and in hypocotyl epidermal cells in the *DUF642* mutants is consistent with the corresponding phenotype. However, a direct interaction of DUF642 proteins with PMEs needs to be established using different experimental approaches. Several proteomic studies have indicated the presence of two isoforms of the same DUF642 protein, suggesting these proteins undergo post-translational modifications that may be related to differential interactions within the cell wall [29,60,61].

The different functions of *DUF642* genes during development can be determined by the specific spatial-temporal expression pattern for each gene. The differential response to hormones also may participate in this process. *BDX* is the only *DUF642* gene expressed in the endosperm during seed development. This specific expression may explain the misshapen-seeds phenotype in the *bdx-1* mutant. Similarly, the expression of *DGR2* in the root elongation zone may explain the short root phenotype exclusive of the *dgr2* mutant. It is possible that there is functional redundancy during embryo development as well as during the development and emergence of the lateral root, processes in which the same temporal-spatial pattern of at least two *DUF642* genes was detected in Arabidopsis.

*BDX*, *At5g11420*, *TEB*, and *At3g08030* are expressed in the meristematic zone of the primary root, but the *bdx* and *teb* mutants do not show an altered root phenotype. BDX was located intracellularly in the epidermal cells of the primary root and relocated to the cell wall in response to biotic and abiotic stresses. Roots interact with different organisms in the soil and are subjected to multiple perturbations in the environment, such as decreases in the availability of water and increases in toxic ions. These interactions can alter the expression of some DUF642 genes. In the response to fungal infection, the overexpression of *VqDUF642* increased resistance to the pathogen, although the expression other *DUF642* genes decreased in response to infection. These results suggest that the *DUF642* genes participate broadly in plant responses to environmental factors and not solely in the root developmental process.

The primary structure of *DUF642* proteins is highly conserved in different spermatophyte species. However, studies of their expression patterns in Arabidopsis showed that the spatial-temporal expression pattern for each gene was specific and consistent with the phenotypes of the mutant plants studied so far. The regulation of *DUF642* gene expression by hormones and environmental stimuli also was specific for each gene. Functional studies of the *DUF642* genes in different plant species are needed to determine the relevance of the DUF642 family in the evolution of terrestrial plants.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/13/3333/s1. Figure S1. *At3g08030* expression during *Arabidopsis thaliana* development using pAt3g08030::ER-GFP plants; Figure S2. *At5g11420* expression during *Arabidopsis thaliana* development using pAt5g11420::ER-GFP plants; Figure S3. *At4g32460/BDX*, *At5g25460/DGR2*, *At5g11420*, *At3g14310/PME3*, *At2g41800/TEB* and *At3g08030* expression during seed germination; Figure S4. Subcellular localization of BDX in *Arabidopsis thaliana* primary roots under salinity stress conditions using pBDX::BDX-GFP plants. Table S1. Transcriptomic evidence of *DUF642* genes expression during specific developmental stages and/or in response to diverse environmental stimuli. Table S2. Transcriptomic evidence of *DUF642* genes expression in response to diverse environmental stimuli.

**Author Contributions:** J.E.C.V., X.G.-M., and A.G.-d.; participated in the conceptualization and in the review and edition of the manuscript. J.E.C.V.; A.S.-I., E.Z.-S., A.H.-B., and E.Q.-R. carried out the molecular genetic studies. J.E.C.-V., X.G.-M., A.S.-I., E.Z.-S., and A.G.-d. participated in the original draft preparation. Funding was provided to A.G.-d. All authors read and approved the final manuscript.

**Funding:** This research was funded by Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de México grant number IN203218- X.G.-M received a fellowship from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México.
**Acknowledgments:** We thank K. Jiménez for assistance on Confocal studies and M. Guemez for the artwork (Figure 1). We thank M. Biswas, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

**Conflicts of Interest:** No conflict of interest is declared.

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