Mechanisms of Water Transport Mediated by PIP Aquaporins and Their Regulation Via Phosphorylation Events Under Salinity Stress in Barley Roots

Abstract

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.

Exogenous application of abscisic acid (ABA) increases root and cell hydraulic conductivity and abundance of some aquaporin isoforms in the ABA-deficient barley mutant Az34

“…The specificity of antibodies used to detect HvPIP2;1 and HvPIP2;2 has been shown previously (Horie et al, 2011).…”

“…Polyclonal antibodies for HvPIP2s were raised in rabbits against synthetic oligopeptides (Medical & Biological Laboratories Co., Tokyo, Japan) corresponding to the amino acid sequences in the N-region of HvPIP2;1 (Katsuhara et al, 2002), HvPIP2;2 (Horie et al, 2011) and HvPIP2;5 (EVMETGGGGDFAAKD, in the present study).…”

“…Root cortex Lp Cell was also analysed in Az34 using the cell pressure probe. We used antibodies raised against ABA (Fricke et al, 2004, Akhiyarova et al, 2006 and anti-(HvPIP2) AQPs antibodies raised specifically against those plasma membrane-localized barley AQP isoforms (HvPIP2;1, HvPIP2;2, HvPIP2;5) which are candidates to facilitate water flow through barley roots (Horie et al, 2011;Knipfer et al, 2011). Immunohistochemistry was used to determine spatial correlations between AQP abundance and local ABA concentrations.…”

Effects of exogenous 5-aminolevulinic acid on PIP1 and NIP aquaporin gene expression in seedlings of cucumber cultivars subjected to salinity stress

“…PIPs have been shown to be primary channels mediating water uptake in plant cells. HvPIP2 has been shown to transport water and have the ability to mediate water loss from plant cells under salt stress (Horie et al, 2011). NIPs only exist in plants and can transport different substrates, not only water, glycerol (Dean et al, 1999), and lactic acid (Choi and Roberts, 2007) but also large solutes such as urea (Wallace and Roberts, 2005), boron, silicon, and arsenic (Mitani-Ueno et al, 2011).…”

Heteromerization of PIP aquaporins affects their intrinsic permeability

“…Antisense inhibition experiments on Arabidopsis thaliana PIP1 and PIP2 have suggested that the two subgroups of aquaporins contribute to root or leaf hydraulic conductivity in the same way (13). In several plant species, members of the PIP1 and PIP2 subgroups were shown to be coexpressed in the same cell type (14)(15)(16)(17).…”