Supporting Information
SI Results and Discussion
Choice of the amino acids used to change the spacing between two NPA domains
Several criteria were used to select the amino acids (AA) to be added or removed from tomato
(SlNIP2-1) and poplar (PtNIP2-1) Si transporters. Protein sequences of proven functional Si
transporters from diverse plant species, from both monocots and dicots, were aligned
(Supplemental Figure 3). The extracellular loop (loop C) was identified as the least conserved
segment therefore the preferred region for mutagenesis experiments. To decrease spacing from
109 to 108 in tomato SlNIP2-1, valine was chosen as it is aliphatic, non-polar and uncharged,
and the smallest among the amino acids present in the less conserved region in loop C
(Supplemental Figure 3). Given that a valine residue at this position is only present in one of the
known Si transporters (V153 in OsLsi1), we reasoned that the identity of the amino acid at this
position was not critical and that an effect associated with its removal would likely be due to the
changed spacing between NPA domains. In the case of Poplar PtNIP2-1, a glycine was added to
increase spacing between the two NPA domains from 108 to 109 AAs for two main reasons: 1) a
glycine residue can fit into hydrophilic or hydrophobic environments, and 2) after addition of a
glycine (G139), the resultant sequence became similar to the sequence present in the functional
Si transporter from soybean. Similarly, a glycine (G139) was removed from the wild-type
PtNIP2-1 sequence to obtain a spacing of 107 AAs between the two NPA domains.
Mutagenesis of the tomato and poplar genes demonstrated that an aquaporin could gain
or lose Si transporting ability by changing the spacing between two NPA domains by as little as
a single AA. Loss of aquaporin function by changing a single AA has been demonstrated
previously, but, the functional gain of an aquaporin through removal of a single AA has, to the
best of our knowledge, never been reported before.
Distribution of aquaporins in plants
Extensive analysis of aquaporins from primitive to higher plants performed here will be
helpful to understand the evolution and specificity of the gene family and transport mechanisms
in plants. For instance, we were able to highlight the evolution of all five sub-families of
aquaporins in primitive plants. This result is surprising since primitive plants, having a simpler
water transport system, harbor nonetheless a complex organization of five aquaporin subfamilies. However, the number of aquaporin genes was found to be lower in primitive plants (see
Fig. 1). As a matter of fact, plant species known to have evolved through recent genomic
duplications bore a higher number of aquaporins. For instance, soybean, carrying 72 aquaporin
genes, has evolved through two separate polyploidy events (Roulin et al., 2013). Most of the
duplicated genes in soybean were found to exhibit sub-functionalization in their expression while
very few have been neo-functionalized or non-functionalized (Roulin et al., 2013). This can be
correlated with the evolution of diverse sub-functionalized expressions of sub-groups in
aquaporins (Ma, 2010; Deshmukh et al., 2013).
Annotation, classification and nomenclature of aquaporins
The enclosed annotated list of 985 aquaporins from 25 plant species can serve as an
extensive reference base for the identification and characterization of aquaporins in many other
plant species. Given that most sequenced plant genomes have been annotated on the basis of
information available in Arabidopsis or rice, this may have prevented the annotation of genes for
which no homologs are found in those two species. This is particularly limiting for aquaporins of
the sub-family XIP and the NIP-III subgroup that are missing in rice and Arabidopsis,
respectively. This is also apparent for the NIPs that have been grouped into varying numbers of
sub-groups in different species (Danielson and Johanson, 2008; Deshmukh et al., 2013). Our
phylogenetic analysis of 260 NIPs has identified three major distinct groups that will facilitate
the classification of future NIPs on the basis of an exhaustive background.
Supplementary References
Danielson, J.A., and Johanson, U. (2008). Unexpected complexity of the aquaporin gene family in the
moss Physcomitrella patens. BMC Plant Biology 8, 45.
Deshmukh, R.K., Vivancos, J., Guérin, V., Sonah, H., Labbé, C., Belzile, F., and Bélanger, R.R. (2013).
Identification and functional characterization of silicon transporters in soybean using
comparative genomics of major intrinsic proteins in Arabidopsis and rice. Plant Molecular
Biology 83, 303-315.
Ma, J.F. (2010). Silicon transporters in higher plants. In MIPs and their Role in the Exchange of
Metalloids (Springer), pp. 99-109.
Roulin, A., Auer, P.L., Libault, M., Schlueter, J., Farmer, A., May, G., Stacey, G., Doerge, R.W., and
Jackson, S.A. (2013). The fate of duplicated genes in a polyploid plant genome. The Plant Journal
73, 143-153.