Kinetic analyses of plant water relocation using deuterium as tracer – reduced water flux of Arabidopsis pip2 aquaporin knockout mutants

Abstract

Due to reduced evaporation and diffusion of water molecules containing heavier isotopes, leaf water possesses an elevated (18)O or (2)H steady-state content. This enrichment has been exploited in plant physiology and ecology to assess transpiration and leaf water relations. In contrast to these studies, in this work the (2)H content of the medium of hydroponically grown Arabidopsis thaliana was artificially raised, and the kinetics of (2)H increase in the aerial parts recorded during a short phase of 6-8 h, until a new equilibrium at a higher level was reached. A basic version of the enrichment models was modified to establish an equation that could be fitted to measured leaf (2)H content during uptake kinetics. The fitting parameters allowed estimation of the relative water flux q(leaf) into the Arabidopsis rosette. This approach is quasi-non-invasive, since plants are not manipulated during the uptake process, and therefore, offers a new tool for integrated analysis of plant water relations. The deuterium tracer method was employed to assess water relocation in Arabidopsis pip2;1 and pip2;2 aquaporin knockout plants. In both cases, q(leaf) was significantly reduced by about 20%. The organ and cellular expression patterns of both genes imply that changes in root hydraulic conductivity, as previously demonstrated for pip2;2 mutants, and leaf water uptake and distribution contributed in an integrated fashion to this reduced flux in intact plants.

Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation

“…These data suggest a model whereby the stomatal closing response to ABA involves an increase in guard cell water permeability mediated by PIP2;1. This function adds to previously described roles of PIP2;1 in root and leaf vein water transport (Da Ines et al, 2010;Péret et al, 2012;Prado et al, 2013) and lateral root emergence (Péret et al, 2012).…”

“…The pip2;1-1 and pip2;1-2 knockout mutants and the two complemented lines, pip2;1-1 PIP2;1 and d35S:PIP2;1ko, were described elsewhere (Da Ines et al, 2010;Péret et al, 2012;Prado et al, 2013). The former is a pip2;1-1 mutant allele transformed with the PIP2;1 genomic sequence (Péret et al, 2012); the latter is a pip2;1-2 mutant overexpressing a PIP2;1 cDNA under the control of a double enhanced cauliflower mosaic virus 35S promoter (Prado et al, 2013).…”

Regulation of Arabidopsis Leaf Hydraulics Involves Light-Dependent Phosphorylation of Aquaporins in Veins

“…Each of these PIPs ranks among the three most highly expressed isoforms in at least one previous gene or protein expression study of Arabidopsis leaves (Alexandersson et al, 2005;Monneuse et al, 2011). T-DNA insertion lines corresponding to knockouts for each of these PIPs in a Columbia-0 (Col-0) (PIP1;2, PIP2;1, and PIP2;6) or Landsberg erecta (Ler) (PIP2;7) background were available from previous studies (Da Ines et al, 2010;Postaire et al, 2010;Péret et al, 2012) or were molecularly characterized in this work (see Supplemental Figure 2 online). To evaluate Arabidopsis rosette aquaporin functionality, we previously established a method for measuring the K ros of internal rosette tissues.…”

“…The SALK_92140c (pip2;6-1) and SALK_118213c (pip2;6-2) Col-0 T-DNA insertion lines and the CSHL_GT19652 (pip2;7-1) Ler transposon insertion lines were obtained from the Nottingham Arabidopsis Stock Center and the Cold Spring Harbor Laboratory, respectively. Homozygous mutations and gene inactivation were verified by genotyping and RT-PCR (see Supplemental Figures 2 and 3 online) (Da Ines et al, 2010;Postaire et al, 2010;Péret et al, 2012) using the primers indicated in Supplemental Table 2 online. All procedures for plant genotyping were as described (Postaire et al, 2010).…”

“…However, no such regulation was found in bur oak (Quercus macrocarpa) (Voicu et al, 2009), and an aquaporin-independent response of shoot hydraulics to irradiance has been reported in laurel (Laurus nobilis) (Nardini et al, 2010). Combined physiological and genetic studies can be used to dissect the role of individual aquaporin genes in plant water transport (Javot et al, 2003;Da Ines et al, 2010;Postaire et al, 2010;Péret et al, 2012). We previously showed that the hydraulic conductivity of excised Arabidopsis rosettes (K ros ) varies diurnally and is the highest in plants at night due to enhanced aquaporin-mediated transport (Postaire et al, 2010).…”