Effects of abiotic stimuli and the phytohormone ABA on the expression of the aquaporin gene family in maize roots

Major intrinsic proteins (MIPs) are an ancient family that were
found in various bacteria, fungi, amphibians, plants and animals.
They are collectively called aquaporins (AQPs) because some members
were found to transport water, and others were shown to be
permeable to small uncharged molecules such as glycerol, urea,
ammonium, or formamide. In the crop plant maize, 34 cDNA sequences
encoding putative AQPs had been previously identified. They include
13 plasma membrane intrinsic proteins (ZmPIPs), 13 tonoplast
intrinsic proteins (ZmTIPs), 5 NOD26-like intrinsic proteins
(ZmNIPs) and 3 small basic intrinsic proteins (ZmSIPs).
Environmental cues such as increased salt concentration or nutrient
deprivation affect root hydraulic conductivity. To examine an
implication of AQPs in these processes, the expression of the maize
MIP gene family was analyzed by a ZmMIP DNA array that harbored
gene-specific sequences as targets for hybridization. Eleven ZmPIP
and 5 ZmTIP members were detected in roots of 11 day-old seedlings
grown in hydroponic culture. Members of the other two subgroups
were expressed weakly. ZmPIP1-1, ZmPIP1-5, ZmPIP2-1, ZmPIP2-4,
ZmPIP2-5, ZmPIP2-6, ZmTIP1-1, ZmTIP2- 1, ZmTIP2-2 and ZmTIP2-4 had
the high abundance. Six day-old maize primary roots were further
dissected to investigate the spatial expression pattern. High
abundance of all detected ZmMIP members were observed in the
elongation zone (3-10 mm) and root hair zone (10-20 mm), but less
transcripts were detected in the root tip (0-3 mm) and mature zone
(50-60 mm). Thus, the expression of aquaporins correlated with the
region of enhanced water uptake. Analysis of aquaporin expression
of longitudinally separated primary roots showed that higher
transcription of ZmPIP1-1, ZmPIP1-5, ZmPIP2-1, ZmPIP2-4, ZmPIP2-5,
ZmTIP1-1, ZmTIP2-2 and ZmTIP2-4 in cortex than in stele tissue.
There was no difference of ZmPIP1-2 and ZmPIP2-6 transcript between
cortex and stele. This indicated that aquaporin-mediated cellular
pathway through cortex tissue played an important role for root
water transport involving the identified isoforms. Maize seedlings
responded differentially to 100 vs. 200 mM NaCl treatments. Leaf
water content was rapidly and persistently reduced after
application of 200 mM NaCl in contrast to 100 mM NaCl. The stress
hormone abscisic acid (ABA) strongly accumulated in roots after 2
h; it stayed at a highly elevated level for 48 h after addition of
200 mM NaCl, but ABA rapidly declined close to control levels in
plants treated with 100 mM NaCl. The transcriptional responses of
ZmMIP genes were also different in both szenarios. Two hours after
addition of 100 mM, but not of 200 mM NaCl, two highly expressed
plasma membrane isoforms ZmPIP1-1 and ZmPIP2-4 were transiently
induced. In addition, the most highly expressed maize PIP gene in
roots, ZmPIP1-5, showed a weak, yet significant induction. None of
the ZmTIP genes was altered. The specific and transient induction
was interpreted as a measure to enhance water uptake when plants
were just regaining the capability of water uptake. In contrast,
multiple ZmPIP and ZmTIP genes were specifically E. Summary 95
reduced by 200 mM NaCl after 24 h. After 48 h, deregulations were
overridden in both cases indicating homeostasis. One µM ABA
exogenously applied to the roots transiently induced ZmPIP2-4
similar to 100 mM NaCl as well as ZmPIP1-2. Previously, an increase
in hydraulic conductivity had been observed upon ABA application.
One hundred µM ABA, a high concentration frequently used in other
studies, lead to a complete, possibly non-specific repression of
all detected ZmPIP and ZmTIP genes after 24 h. It is inferred that
the early induction of ZmPIP2-4 by NaCl may be mediated by ABA,
whereas other regulations are probably ABA-independent.
Furthermore, transcriptional repression of aquaporins could be
involved in the reduction of water permeability after treatment
with 200 mM NaCl, but not after milder salt stress. Aquaporin
abundance was also investigated at the protein level in total
microsomal fractions using antisera detecting ZmPIP2-3 and ZmPIP2-4
as well as the ZmPIP1 subgroup. Apparently, the protein level of
aquaporins was not correlated to the transient changes in mRNA
abundance. This may point to a real discrepancy of mRNA and protein
levels. Alternatively, other detected isoforms, which were not
altered, might obviate the resolution of changes, or a possible
membrane translocation was not resolved when using microsomal
fractions. Phosphorylation of aquaporins has been reported as
another possible mechanism to regulate aquaporins. However, with
respect to NaCl and ABA treatments no alterations in the degree of
phosphorylation could be detected in this study, which, however,
could not discriminate between different isoforms. Finally, the
implication of AQPs in changes of hydraulic conductivity provoked
by K+- and NO3 --deprivation was examined in a preliminary study.
K+-deficiency reduced four ZmPIP and three ZmTIP members. This
could be related to a reduced cellular water transport through
roots as well as the retarded growth although overall water flux
was increased. Surprisingly, ZmPIP1-5 and ZmTIP1-1 was specifically
repressed after N-deprivation. This change may indicate that these
isoforms are primarily involved in the transcellular water flow
that is reduced under N-limitation; yet at the same time enhanced
root growth is possible, which involves other isoforms that were
not affected in this instance.