5. Discussion
5.3 Old players, new tricks
The transition from the aquatic environment to land implicated the development of several adaptations to a dry environment. Among them is the appearance of several hydrophobic polymers, such as lignin, cutin, or suberin, that confer protection from pathogens and against dissection of the terrestrial organisms’ tissues. Algae, Non-Vascular Plants and Vascular Plants share common ancestors, thus the comparative analysis of their genomes and protein structures might aid in understanding the appearance of the suberin biosynthetic and regulatory machinery during evolution. By analysing the candidate proteins domains and the presence/absence across Algae and Land plants we expected to learn more about these proteins putative function during suberization. The fact that most proteins domains are quite conserved among all species suggests that these proteins were conserved during land colonization. The ANT protein in Arabidopsis has only one identified domain, APETALA2 (AP2), that was found in all species. Interestingly, a domain that is predominantly present in proteins that are involved in floral meristem identity and seed development (Luo et al., 2021) was found in Klebsormidium nitens, suggesting that an ANT-like transcriptional factor could regulate other physiologic processes in the early stages of plant land colonization. AP2 Domain was also found in non-vascular plants, more specifically in Physcomitrella patens and Marchantia polymorpha. Since “seed” and “flower” are structures that are not found in this major group, the previous idea that this domain was involved in different processes during Land colonization is once again reinforced. In Aoyama et al. (2012), this hypothesis is corroborated when four AP2-type transcription factors, orthologs to AINTEGUMENTA, were considered indispensable to the formation of gametophore apical cells from protonema cells of Physcomitrella patens (Aoyama et al., 2012).
ATHB6 protein, in Arabidopsis, is composed by two HOMEOBOX domains present in all species (Figure 4.3). These two domains were found in the Algae group, however, considering the redundancy of the HOMEOBOX domain and the phylogenetic distance between Klebsormidium nitens and the species from the vascular plant group, probably the protein sequence found in the Algae group participate in a different biological process. Interestingly, in Physcomitrella patens, the top hit sequence found (BAC54164) was analysed in Sakakibara et al. (2007) and was inferred that the correspondent gene, PPHB7, was involved in epidermal cell differentiation, since mutants reveal the induction of pigmentation and the increase in the size and number of chloroplasts. When comparing with Arabidopsis, the idea of a similar molecular function that regulate different biological processes is reinforced.
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MYB36 encodes a transcriptional factor, whose two SANT domains promote chromatin remodelling and transcription regulation. All species from the three major groups possess this domain in the top hit sequences. The role of MYB protein super family in Algae species is still poorly understood. Around 50 MYB transcriptional factors were identified in green algae and current literature suggests that this family underwent a large-scale expansion in bryophytes group (Non-Vascular Plants), more specifically in Physcomitrella patens, where more than 100 MYB genes have been identified (Pu et al., 2020). The expansion of this regulatory gene family may have occurred in response to new abiotic stresses when plants started colonizing the land. This hypothesis is also corroborated by the fact that the expansion of other regulatory gene families in plants is greater when compared with animals, as result of more recent genome duplications (Feller et al., 2010). In Arabidopsis, MYB36 is associated with drought stress response (Liu et al., 2022), suggesting, that this protective mechanism could result, in part, from MYB gene family expansion in the bryophyte group.
RAX3 belongs, also, to the MYB transcriptional factor family, however, this gene is less understood in current literature, when compared to MYB36. According to NCBI database, this gene in A. thaliana has four MYB-like ortholog genes (A0A2K1LC00, A0A2K1JR42, A0A2K1JYR9 and A0A2K1KV94) in Physcomitrella patens, however a deeper analysis of this sequences was not performed.
CLV1, in Arabidopsis, is composed by two distinguish domains, Leucine Rich Repeat (LRR) and Protein Tyrosine and Serine Kinase, that were found in all vascular and non-vascular species however the second domain was not found in the top hit sequence from Algae Group (Figure 4.3 A).
Literature suggests that CLV1 is absent from algae, however the top hit sequence obtained (Figure 4.3 A) in Klebsormidium nitens, also known as Klebsormidium flaccidum, was analysed by Hori et al (2014, that demonstrated K. flaccidum genome encodes putative variants of functional receptors and transporters found in land plants. The absence of the protein kinase domain in Klebsormidium flaccidum top hit sequence (Figure 4.3 A) suggests that the biological function of AtCLV1 was not conserved in algae and it was a response to the environmental challenges imposed during terrestrial adaptation.
WOX9 protein, composed by one homeobox domain in Arabidopsis, has a crucial role in maintaining stem cell identity in the shoot of vascular plants. In Lian et al. (2014) phylogenetic analysis inferred that WOX proteins present in green algae represented the oldest members of WOX protein family and form an ancient clade (Figure 5.1 A). The clade was composed by green algae and bryophytes, however, WOX9 was only found in an intermediate clade that, according to authors, contained all the vascular plants (Figure 5.1 B). This explains the clusters obtained in the phylogenetic tree (Figure 4.3 B), since WOX9 protein seems to be present only in vascular plants and so its structure is more conserved among this group. This also suggests that the WOX9 was not generated by the great genomic expansion that occurred in the bryophyte group, in response to a new environment.
The genomic analysis suggests thatWOX9 first appeared in the Lycopodiophytes and were kept conserved throughout time. According to this data, the top hit sequences obtained in Klebsormidium nitens, Marchantia polymorpha and Physcomitrella patens (Figure 5.1 B) correspond to WOX9-like proteins that belong to an ancient clade and most likely have a different function. The biological process that is regulated by WOX9 transcriptional factor seems to emerge much later, during plant land colonization. Since this gene was first noticed in the intermediate clade, it would be interesting to include more species from Gymnosperms and Lycopodiophytes to understand more about the evolutionary origin of WOX9 and its role.
To further elucidate the evolutionary history of WOX9 a motif scan was performed and, the homeobox domain was found in all species except the Algae group. Interestingly, motif 1 was found in
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bryophytes and vascular plants (Figure 4.3 C Blue Star). This motif in Marchantia polymorpha and Physcomitrella patens may have had a different function despite the similar domain structure. In Arabidopsis and Brassica rapa, two additional HOMEOBOX like motifs were found (motif 8 and 10) (Figure 4.3 C Red Star).
Figure 5.1. WOX9 Evolution Throughout Land Colonization. (A) Diagram representing a proposed model by Lian et al.
(2014) to explain the evolutionary history of WOX family. The schematic tree represents the different taxa that mark plant evolution and associate each one to three clades found in WOX protein family (Ancient, Intermediate and WUS). (B) Phylogenetic tree obtained in the present study, and proposed clades. It is possible to observe that the bryophyte group and the green algae group form a single cluster and the remaining angiosperms form another clade. Abbreviations: Bry: Bryophyte, Lyc: Lycopodiophyte, Gym: Gymnosperm, Mon: Monocots, Eud: Eudicots, Ppa: Physcomitrella patens, Smo: Selaginella moellendorffii, Pta: Pinus taeda, Osa: Oryza sativa, and Ath: Arabidopsis. Image adapted from Lian et al. (2014).
This may suggest that this protein could have suffered an evolutive divergence event in the Brassicales order that is also supported by the clade formed by these two species in the phylogenetic tree (Figure 5.1 B). Since these two motifs were not found in Quercus suber, WOX9 protein in Arabidopsis may regulate other biological roles. To further explore these motifs it would be interesting to do a phylogenetic analysis of WOX9 among species from the Brassicales order.
5.4 Artefacts, or something more?
During the image analysis to the suberization patterns in myb36-1 roots, as acquired at the fluorescence microscope, the transition zone between the non-suberized and patchy areas in 10 out of 11 seedlings, seemed to have different progression properties from all other genotypes. In other mutants and corresponding wild type controls, the patchy zone started usually with a single suberized endodermal cell. In myb36-1, however, it was not possible to individualize a starter cell, in fact, a “fade-like”
suberization pattern was usually observed (Figure 5.2 A). This phenotype was not observed or described in any similar histological analysis of myb36-1 (Wang et al., 2020). The hypothesis that this alteration is a histological artefact could be considered, however, all seedlings (myb36-1 and Col-0) were submitted to the same plating and staining procedures and conditions. This alteration in early suberized structures and the high percentage of suberized endoderm cells in the continuous zone in the loss of
25
function mutant reinforce the idea of a putative involvement of MYB36 in suberin deposition along the root axis. At first sight, another artefact seems to be present in the continuous zone of all genotypes analysed.
Figure 5.2 Alterations in non-suberized-patchy transition zone patterns and passage cells. (A) The transition zone showing the “fade” like suberization pattern observed in most myb36-1 seedlings versus the wild-type pattern observed in Col-0. (B) Continuous zone of wild-type genotypes and the “passage cells”, that are highlighted with white arrows. In (A) and (B) the scale bar corresponds to 100 µm.
Single-non-suberized endoderm cells were found in this zone, where all cells should be fully suberized (Figure 5.2 B). Current literature calls these structures “passage-cells” (Holbein et al., 2021 and Andersen et al., 2015) and their function is still not completely understood. The absorption of solutes from the soil is achieved through radial transport to the vascular cylinder. In older suberized root parts, the continuous ring of fully suberized and lignified cells hinders this transport. The only structure that could allow this transcellular transport would be the passage cells (Holbein et al., 2021). It is also speculated that these cells could allow the establishment of symbiotic relationships with other organisms, like plant-microbes or plant-fungi interactions, that could facilitate nutrient uptake and provide biotic and abiotic protection (Holbein et al., 2021). To further corroborate the involvement of WOX9 in the biosynthesis and deposition of suberin, it would be interesting to do an expression analysis of this gene in this non-suberized cell through a reporter line.
6. Conclusion
The present work analysed the involvement of ANT, ATHB6, CLV1, MYB36, RAX3 and WOX9 genes, found in cork secondary growth tissues, in the process of cork oak trunk suberization. Using Arabidopsis as an alternative plant model, the impact of these genes was assessed marking the respective mutants with a suberin molecular marker and analysing the patterns of suberin deposition. ant-9, myb36-1, stip-1, stip-2 and stip-D revealed robust alterations in suberization patterns, suggesting that these could act as positive regulators of suberin pathway/signalling, except myb36-1 that exhibit an increase in suberin deposition. athb6, clv1-1 and rax3-1 did not show significative differences, however, these could influence suberin composition without affecting its biosynthesis/ deposition and a further suberin chemical analysis should be performed in these genotypes.
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Since WOX9 displayed major differences in the histological assays, the expression analysis of suberin-related genes in stip-1 and stip-D mutants found KCS2, CYP86A1, FAR1, CYP86B1 and GPAT5 genes from aliphatic pathway to be altered. ASFT, a gene involved in the phenylpropanoid pathway, and genes involved in the transport of suberin building blocks (ABCG6) and in suberin lamella polymerization machinery (GELP38) also showed reduced expression in mutants. The suberin patterns observed in the histological assay suggest that other suberin-related genes, should be analysed for expression, more specifically nuclear transcriptional factors. The ABA-induced rescue of stip-1 mutants suggests that WOX9 is upstream of ABA and involved in endoderm suberization, in early stages of Arabidopsis root development. Current literature suggests that this suberization could occur through ABA-mediated GPAT5 upregulation (Barberon et al., 2016). Ongoing work in the host lab will help elucidate putative interactions between WOX9 and ABA signalling/biosynthesis/catabolism-related genes.
During plant development, WOX9 seems to act as a positive regulator of the suberin pathway and we speculate could influence it through a putative WRKY9 upregulation, inducing consequently CYP86B1 activity. The existence of relationship between WOX9 and WRKY9 should be elucidated in further work. However, this theory is only a putative piece of a complex signalling network that remains fairly unknown. Future work on possible protein-protein interactions, or WOX9-mediated regulated genes should be performed, including in silico analysis searching for WOX9 TF-binding sites to promoter regions in genes known to belong to the suberin regulatory and/or biosynthetic pathway, and then validating experimentally by in vitro protein-protein and DNA-protein binding assays, for instance.
The phylogenetic analysis involving ANT, ATHB6, CLV1, MYB36, RAX3 and WOX9 proteins reveal interesting aspects about their involvement in land colonization. ANT, ATHB6, RAX3, MYB36 and WOX9 appear to be well conserved throughout land colonization, regulating some developmental processes that most likely were present in organisms from both aquatic and terrestrial environments. CLV1 seems to be the only gene that result from an adaption response to a new terrestrial environment. A deeper analysis about evolutionary history of WOX9 revealed that this gene is present only in vascular plants, despite the fact that its family is present in green Algae. The WOX9 protein structure among Brassicales order suggest that this transcriptional factor could regulate other biological processes in species that belong to this taxon, and further studies should aim to explore this difference and demystify more unknown concepts about WOX9 evolutionary history.
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31 8. Supplementary information
8.1 Suberin staining Protocol
1. Vertically grown (in MS-agar plates) 7-days old seedlings are incubated in a freshly prepared solution of Fluorol Yellow 088 (0.01%w/v, in lactic acid) at 70°C for 30 min.
2. Transfer fresh seedlings with similar length to histological cassettes.
3. Rinse in water (three baths of 3 min each).
4. Counter-stain with aniline blue (0.5% w/v, in water) at room temperature for 15 min in the darkness. Stir gently every 5 minutes.
5. Wash in water for, at least, 30min (change the bath to fresh water every 5 minutes). Keep the seedlings in the darkness.
6. Transfer the seedlings to a glycerol 25% bath to preserve tissue integrity.
7. Mount using Anti-fade medium prior to microscope examination. Handle the seedlings gently, to preserve the tissues, and mount them as straight as possible to facilitate prior observation.
Remarks and tips
• Always use a freshly prepared solution of Fluorol Yellow.
• Aniline blue can be stored, at room temperature at obscurity, and used again in further stains.
• When transferring the seedlings to the histological cassettes be relatively agile to not let them dry.
Microscopy
• Use a wide-field microscope with a standard GFP filter to observe Fluorol Yellow and counterstain.
Remarks
• After staining, keep samples in the dark at cold temperature (4 to 6 ºC).
• Do not use samples 3 hours after preparation, as the fluorescent signal may leak into the xylem.
• Do not keep the seedlings under fluorescence for long periods since Fluorol Yellow is easily bleached.
Adapted from:
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