**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 1.** Expression patterns of the *DUF642* genes described to date using reporter genes during Arabidopsis plant development.

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 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.

**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.

#### **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 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].

#### **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 *AhDGR2* 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 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.
