Identification and expression of nine oak aquaporin genes in the primary root axis of two oak species, Quercus petraea and Quercus robur

Abstract

Aquaporins (AQPs) belong to the Major Intrinsic Protein family that conducts water and other small solutes across biological membranes. This study aimed to identify and characterize AQP genes in the primary root axis of two oak species, Quercus petraea and Quercus robur. Nine putative AQP genes were cloned, and their expression was profiled in different developmental root zones by real-time PCR. A detailed examination of the predicted amino acid sequences and subsequent phylogenetic analysis showed that the isolated AQPs could be divided into two subfamilies, which included six plasma membrane intrinsic proteins (PIPs) and three tonoplast intrinsic proteins (TIPs). We characterized the anatomical features of the roots and defined three developmental root zones: the immature, transition and mature zones. Expression analysis of the AQPs was performed according to these root developmental stages. Our results showed that the expression of PIP2;3 and TIP1 was significantly higher in Quercus petraea compared with Quercus robur in the three root zones. However, PIP2;1 and TIP2;1 were found to be differentially expressed in the mature zone of the two oak species. Of the nine AQP genes identified and analyzed, we highlighted four genes that might facilitate a deeper understanding of how these two closely related tree species adapted to different environments.

Figure 1. Anatomical features of Quercus robur and Quercus petraea primary roots.

(A) Localization of apoplastic barriers along the primary root axis of five-week-old oak seedlings. Vibratome cross-sections through primary roots of Quercus robur at 1 cm (g,h), 2 cm (d,e) and 3 cm (a,b) from the root tip stained with berberin hemisulfate. (c,f,i) Detailed view of endodermis differenciation for Quercus petraea, at different distance from the root tip. (h,i) Sections through immature zone display discrete dots of fluorescence in the radial walls of endodermal cells (white arrow) which reveals insignificant suberin deposits. (e,f) In the transition zone, the suberization of some endodermal cells (en) was observed. (b,c) Sections cut at 3 cm from the root tip revealed a complete ring of suberized endodermal cells (en). A suberized exodermis (ex) was detected in the three section levels. Scale bars: 100 µm. (B) Localization of the zone of cell elongation in oak root tip. (a) A longitudinal root section in immature root zone of Quercus robur stained with hematoxylin to measure the length of cortical cells in the root tip. ctx: cortex, rcj: root cap junction. Scale bar: 100 µm. (b) Length of cortical cells as function of distance from the root tip in both oak species. Means ± standard error of the mean (SEM) (n = 4), for each oak species.

Figure 2. Phylogenetic analysis of oak AQP proteins.

Phylogenic tree showing the four clusters PIP1, PIP2, TIP1 and TIP2. The nine Quercus petraea AQPs are compared with all the PIPs as well as all the TIP1s and TIP2s from Arabidopsis thaliana and Populus trichocarpa. Maximum likehood phylogenetic analysis and bootstrap test were performed using MEGA 5. Identified subgroups are indicated by different colors and oak AQP names are marked by full circles. Branch lengths are proportional to evolutionary distance.

Figure 3. Comparative alignment of predicted amino acid sequences isolated from Quercus petraea and representative AQPs in plants.

The four AQPs SoPIP2;1 (AAA99274.2), RsPIP1;3 (BAA92259.1), VvTnTIP2;1 and ZmTIP1;1 (NP_001104896.1) have been functionally characterized as water channels. Black boxes represent predicted transmembrane helices and the AQP NPA sequence signature is underlined in black. The conserved amino acid typically found in the constriction region of the pore (Ar/R filter) are indicated by stars, and conserved residues located at Froger’s positions are shaded in blue. Amino acids identified for being involved in specificity of water transport are shaded in red. Serine residues, appointed by arrows, are a component of plant AQP gating and residues occurring in the helix-helix interfaces are underlined in dark grey.

Figure 4. AQP transcript abundance in primary roots of Quercus robur and Quercus petraea.

Expression analysis was determined from 21 independent RNA preparations. Means ± standard error of the mean (SEM) (n = 21).

Figure 5. AQP transcript abundance in the developmental primary root zones of Quercus robur and Quercus petraea.

The relative expression of AQPs was measured in different root segments from the root tip of the immature, transition, and mature zones. For each root zone, expression analysis was performed from 21 RNA extractions, which corresponded with 21 batches of three oak seedlings. Means ± SEM (n = 21). Significant differences are indicated by the letters a, b and c.