ASSESSMENT OF THE HOMOLOGY OF THE AQUAPORIN IN REPRESENTATIVES OF SYMBIOTIC MYCORRHIZAL FUNGI

Globally, particularly in arid and semi-arid regions, water is the primary environmental factor limiting productivity in terrestrial ecosystems

Arbuscular mycorrhizal (AM) fungi, belonging to the monophyletic phylum Glomeromycota, colonize over 80% of terrestrial plants

Aquaporins constitute major pathways for transmembrane water movement

The proteins AQPF1 and AQPF2 are two important aquaporins in AMF, and these proteins are related to the drought adaptation mechanisms initiated by symbiotic relationships 

Therefore, this study aims to assess the amino acid sequence homology of aquaporins AQPF1 and AQPF2 across mycorrhizal fungi species to advance understanding of "fungus-plant" symbiotic dynamics under drought conditions.

The signaling proteins AQPF1 and AQPF2, identified across diverse arbuscular mycorrhizal fungi (AMF), were selected as research targets. Amino acid sequences for alignment construction were retrieved from the NCBI Protein database, and use the program BLAST to search for homologues of AQPF1 and AQPF2. The AQPF1 and AQPF2 protein sequence of Rhizophagus intraradices (with confirmed gene annotation and chromosomal localization) served as the initial query. Homologs of AQPF1 and AQPF2 were identified using the BLASTP program

1) Rhizophagus intraradices (AFK93202.1);

2) Rhizophagus diaphanus MUCL43196 (RGB28923.1);

3) Rhizophagus irregularis (PKC58620.1);

4) Rhizophagus clarus (BAU71502.1);

5) Acaulospora morrowiae (CAG8589232.1);

6) Cetraspora pellucida (CAG8524555.1);

7) Diversispora epigaea (RHZ56822.1);

8) Diversispora eburnea (CAG8432930.1);

9) Dentiscutata erythropus (CAG8473267.1);

10) Dentiscutata heterogama (CAG8685703.1);

11) Funneliformis mosseae (CAG8578260.1);

12) Funneliformis caledonium (CAG8628874.1);

13) Funneliformis geosporum (CAI2164236.1);

14) Glomus cerebriforme (RIA94752.1);

15) Gigaspora margarita (KAF0497703.1);

16) Gigaspora rosea (RIB30586.1);

The species of the AQPF2 gene analyzed is exactly the same as those listed above. However, there are some minor differences in the protein names, which is due to the existence of known amino acid sequence information in the NCBI protein database

1) Rhizophagus intraradices (AFK93203.1);

2) Rhizophagus diaphanus MUCL43196 (RGB43499.1);

3) Rhizophagus irregularis (XP_025170192.1);

4) Rhizophagus clarus (BAU71503.1);

5) Acaulospora morrowiae (CAG8574845.1);

6) Cetraspora pellucida (CAG8528323.1);

7) Diversispora epigaea (RHZ86381.1);

8) Diversispora eburnea (CAG8436721.1);

9) Dentiscutata erythropus (CAG8473267.1);

10) Dentiscutata heterogama (CAG8513861.1);

11) Funneliformis mosseae (CAG8590313.1);

12) Funneliformis caledonium (CAG8445309.1);

13) Funneliformis geosporum (CAI2179106.1);

14) Glomus cerebriforme (RIA98684.1);

15) Gigaspora margarita (CAG8854038.1);

16) Gigaspora rosea (RIB15405.1);

To identify differences between proteins, bioinformatic analysis was performed. It included multiple alignment of the amino acid sequences of the studied proteins using the MEGA 12 program (MEGA, Japan) 

The multiple sequence alignment of AQPF2 revealed high conservation of this aquaporin, with a maximum pairwise genetic distance of 0.30167 observed between Acaulospora morrowiae and Glomus cerebriforme. No significant variable regions were detected at the N-terminus (Fig. 1). While the central region remained largely conserved across species, a 6-amino acid deletion was identified specifically in Diversispora epigaea and D. eburnea (Fig. 2). No other major indels were observed in this region.

Figure 1 - N-terminus of AQPF2 alignment

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Figure 2 - Internal Deletion Region in the alignment of AQPF2 Protein

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At the C-terminus, members of the Gigasporaceae family (Dentiscutata erythropus, D. heterogama, Gigaspora rosea, and G. margarita) exhibit a conserved 14-amino acid deletion (Fig. 3). Adjacent to this deletion, a polymorphic region spanning approximately 16 residues was identified.

Figure 3 - C-terminus of AQPF2 alignment

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The AQPF2 phylogenetic tree broadly mirrors the species-level phylogeny

[17]

. Notably, certain species yielded two distinct protein sequences, annotated as AQPF2. Intriguingly, the genetic distance between these intra-species paralogs (e.g., 0.11778 in Rhizophagus intraradices) exceeded distances to cross-species orthologs (e.g., 0.09270 between R. intraradices and Gigaspora rosea). Nevertheless, all Glomeraceae proteins formed a monophyletic clade (Fig. 4), the pairwise genetic distance threshold for the phylogenetic confidence level was selected to be 0.16, indicating post-familial divergence of this aquaporin.

The proteins that are most distant from the others are Diversispora epigaea and Diversispora eburnea from the Diversispora genus; their branch is longer than all other branches and is classified as a separate evolutionary branch.

Figure 4 - AQPF2 proteins phylogenetic tree

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AQPF1 exhibits significantly reduced sequence conservation relative to AQPF2, featuring an N-terminal variable region (Fig. 5). Maximum pairwise divergence (0.54047) was observed between Rhizophagus clarus and Acaulospora morrowiae.

Figure 5 - N-terminus of AQPF1 alignment

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Beyond discrete polymorphic segments within the internal sequence (Fig. 6), the C-terminal domain harbors an extended variable region spanning up to 50 residues. Notably, Dentiscutata heterogama exhibits a distinct 16-amino acid deletion within this domain (Fig. 7).

Figure 6 - Insertions and variable regions in the alignment of AQPF1 Protein

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Figure 7 - C-terminus of AQPF1 alignment

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The AQPF1 phylogeny groups Gigasporaceae (order Diversisporales) and Dentiscutata heterogama into a long-branched clade (Fig. 8). This is different from the evolutionary structure of species

[17]

. Meanwhile, the protein differentiation of Rhizophagus clarus occurred earlier than the formation of the family (Fig. 8).

Figure 8 - AQPF1 proteins phylogenetic tree

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Compared to AQPF1, AQPF2 displays exceptional conservation. The presence of multiple paralogs in fungal genomes complicates phylogenetic reconstruction, and their functional divergence remains uncharacterized. Conversely, AQPF1 exhibits substantial sequence plasticity, with intra-Glomeromycota genetic distances typically exceeding 0.5. This structural variability positions AQPF1 as a priority target for elucidating AM fungal mechanisms in enhancing plant drought tolerance.

Notably, unlike typical proteins where variation localizes to terminal regions

By comparing the two aquaporin proteins AQPF1 and AQPF2 and constructing a phylogenetic tree, the results revealed the conservation of the AQPF2 protein and the variability of the AQPF1 protein. AQPF1 has been identified as a promising protein for searching for intraspecific variations related to drought resistance.