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Zhu, Chuanfeng (2005): Effects of abiotic stimuli and the phytohormone ABA on the expression of the aquaporin gene family in maize roots. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

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.