This document identifies and analyzes aquaporin genes in barley in response to salt stress. It isolates several new aquaporin genes (HvPIPs) in barley. It finds that under salt stress, transcription of several HvPIP genes decreases more in salt-sensitive barley compared to salt-tolerant barley. HvPIP2;3 and HvPIP2;4 showed high water transport activity, while HvPIP1;2 did not, but coexpression of HvPIP1;2 with HvPIP2;1 or HvPIP2;4 increased water permeability. Downregulation of some PIP genes under salt stress may limit water loss.

1. オオムギ原形質膜型アクアポリ
ン遺伝子の同定と塩ストレスに
よる発現制御
辻 伸弥
( 岡山大･植物研 )

2. Two parameters determine the amount of water
transport.
･ motive force (difference in water potential)
･ water permeabilityWater channels formed by
aquaporins
Water uptake is one of the most basic functions of plant
root systems.
This function is disturbed by salt stress.
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,

3. Barley (Hordeum vulgare)
Salt tolerant cultivars (Haruna-nijo, K305)
Salt sensitive cultivar (I743)
NaCl
(mM)
1 cm 1 cm
K305
0 10
0
20
0
I743
0 10
0
20
0
The effect of NaCl treatment for 24 h on
the growth of 4-day-old seedlings.

4. 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

5. Expression analysis of HvPIP
genes
･ 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.

6. Comparing expression of HvPIP genes among
cultivars
Date are means ± SD
(n=6)
In 4-day-old root

7. Haruna-nijo Effect of 200 mM NaCl on HvPIP genes
expression
Date are means ± SD
(n≧3)

8. K305 Effect of 200 mM NaCl on HvPIP genes
expression
Date are means ± SD
(n≧3)

9. I743 Effect of 200 mM NaCl on HvPIP genes
expression
Date are means ± SD
(n≧3)

10. salt stress
water
plasma
membrane
PIP
･ HvPIP1;2 exhibited the highest transcription in 4-day-old
root.
･ Transcript levels of HvPIP1;2, HvPIP2;1, HvPIP2;2 and
HvPIP2;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 under
high salinity.

11. Functional analysis of HvPIPs expressed in Xenopus
oocytes
Swelling assay (typical
examples)
HvPIP2;3 and HvPIP2;4 have high water channel
activity in Xenopus oocytes, whereas HvPIP1;2 is
inactive.

12. 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