Water uptake is one of the most basic functions of plantroot systems.This function is disturbed by salt stress.Two parameters determine the amount of watertransport. ･ motive force (difference in water potential) ･ water permeability Water channels formed by aquaporins Plant aquaporin PIP (plasma membrane intrinsic protein) TIP (tonoplast intrinsic protein) NIP (nodulin-26 like intrinsic protein) SIP (small basic intrinsic protein) Aquaporins facilitate the diffusion of water across plant cell membranes. Maurel and Chrispeels (2001) Plant Physiol., 125,
Barley ( Hordeum vulgare ) Salt tolerant cultivars (Haruna-nijo, K305) Salt sensitive cultivar (I743) NaCl 0 10 20 0 10 20 (mM) 0 0 0 0 K305 I743 1 cm 1 cm The effect of NaCl treatment for 24 h on the growth of 4-day-old seedlings.
Isolation of HvPIP genes ･ Design PCR primers for contig sequences of HvPIP genes. ･ Perform RT-PCR on total RNA from roots of Haruna-nijo seedlings. Isolated previously HvPIP1;3 , HvPIP1;5 , HvPIP2;1 Katsuhara et al. (2002) Plant Cell Physiol., 43, 885-893 HvPIP2;4 Panda et al. (2005) Published Only in Database Isolated in the present study 6 novel genes HvPIP1;1 , HvPIP1;2 , HvPIP1;4 , HvPIP2;2 , HvPIP2;3 , HvPIP2;5 Full length cDNA HvPIP1;1 , HvPIP1;2 , HvPIP1;4 , HvPIP2;3
Expression analysis of HvPIPgenes ･ Extract total RNA from 4-day-old roots of Haruna-nijo, K305 and I743 treated with salt stress. Salt stress : 100 mM or 200 mM NaCl for 1 h, 2 h, 4 h, 8 h, 12 h, 24 h Control : 0 h and 0 mM NaCl for 1 h, 2 h, 4 h, 8 h, 12 h, 24 h ･ Perform absolute quantitative real time RT-PCR. Except HvPIP1;3 and HvPIP1;4.
Comparing expression of HvPIP genes amongcultivarsIn 4-day-old root Date are means ± SD (n=6)
Haruna-nijo Effect of 200 mM NaCl on HvPIP genes Date are means ± SD (n≧3)expression
K305 Effect of 200 mM NaCl on HvPIP genes Date are means ± SD (n≧3)expression
I743 Effect of 200 mM NaCl on HvPIP genes Date are means ± SD (n≧3)expression
･ HvPIP1;2 exhibited the highest transcription in 4-day-oldroot.･ Transcript levels of HvPIP1;2 , HvPIP2;1 , HvPIP2;2 andHvPIP2;3 were decreased by salt stress (200 mM NaCl).･ It is suggested that water permeability is reduced by down-regulation of PIP genes in order to limit loss of water underhigh salinity. plasma membrane salt stress water PIP
Functional analysis of HvPIPs expressed in Xenopusoocytes Swelling assay (typical examples) HvPIP2;3 and HvPIP2;4 have high water channel activity in Xenopus oocytes, whereas HvPIP1;2 is inactive.
Coexpression of HvPIP1;2 and HvPIP2 isoforms in Xenopus oocytes ･ Coexpression of HvPIP1;2 and HvPIP2;1 or HvPIP2;4 in Xenopus oocytes resulted in an increase in osmotic water permeability. ･ HvPIP1;2 did not interact functionally with HvPIP2;3 in