Study On The Relationship Between Aquaporins And Responses To Drought And Chilling Stresses In Plant

Aquaporins, which show the ability to facilitate passive exchange of water acrossmembranes, belong to a high conserved membrane protein family MIP (Major IntrinsicProtein). In plants, the activities of aquaporins can be controlled at transcription, translationand post-translation modification levels. The discovery of aquaporins in plants has resulted ina paradigm shift in plant physiology.In this study, we focused on the role of rice aquaporins when respond to drought andchilling stress. The main results are as follows:(1) The mechanism of drought tolerance was studied by comparing water relationsbetween an upland rice cultivar (Zhonghan 3) and a lowland rice cultivar (Xiushui 63) duringthe water deficit mediated by PEG. After 7?8 days with osmotic stress treatment, bothcultivars showed retardation of plant growth, dwarf plants and young leaf rolling or evendrying. These symptoms were more serious in Zhonghan 3 than Xiushui 63. The osmoticstress greatly retarded the growth of roots and aerial parts in Zhonghan 3 than Xiushui 63.After initiation of the osmotic stress, relative water content (RWC) and water potential ofleaves decreased significantly in both cultivars, and Xiushiu 63 had higher leaf RWC andwater potential than Zhonghan 3. Xiushui 63 probably showed higher osmotic adjustmentbecause Xiushui 63 had greater drop of leaf osmotic potential and higher turgor compared toZhonghan 3 when subjected to the osmotic stress. After initiation of the osmotic stress,Zhonghan 3 showed decreased transpiration rate and stomata conductivity, while they wereunchanged in Xiushiu 63. Root hydraulic conductivity (Lp) decreased significantly in bothcultivars after initiation of the osmotic stress. Moreover, by view of the freehandcross-sections of adventitious roots under a fluorescent microscope, no any differentlignifications at exodermis or endodermis in the roots of both cultivars was observed.Mercurial compounds are widely used to study the function of aquaporins because they areinhibitors for aquaporins. Mercury treatment greatly decreased root hydraulic conductivity ofnon-stressed plants in both cultivars. However, during the osmotic stress, mercury showedmore serious inhibition effects in Zhonghan 3, while showed alleviating inhibition effects inXiushiu 63. To summarize, all these results suggested that upland rice and lowland mobilizeddistinct mechanism to resist drought, i.e. upland rice was drought avoidance (e.g. increasingroot water uptake and decreasing water evaporation) while lowland rice was drought tolerance(e.g. osmotic adjustment). Aquaporins might play role in drought avoidance of upland rice.(2) To demonstrate the role of aquporins in plant drought avoidance in fine detail,aquaporin RWC3 from rice (Oryza sativa L.) was transferred into a lowland rice cultivars(Zhonghua 11), which was thought to be weak in drought avoidance, and consequently somephysiological parameters were measured during the water deficit mediated by PEG.SWPA2::RWC3 was constructed into a binary plasmid pCAMBIA 1301, and was transferred tolowland rice mediated by Agrobacterium tumefaciens. SWPA2 promoter, which was isolatedin sweetpotato, was a stress inducible promoter. By the assays of GUS histochemical staining,PCR and PCR-Dig-ELISA, the presence of SWPA2::RWC3 was confirmed in the lowland rice.By RT-PCR and Western blot analysis, it was found that SWPA2 promoter-RWC3 transgenicplant and WT (wild type) had similar RWC3 expression levels under normal condition,nevertheless, under water deficit mediated by 20% PEG, the transgenic plant had higherRWC3 expression levels than WT, suggesting that RWC3 expression in transgenic plants couldbe induced by drought. However, no visible phenotypic difference was found between thetransgenic plants and the WT plants, either under normal condition or during water deficit.After the water deficit, SWPA2 promoter-RWC3 transgenic plant showed higher root hydraulicconductivity and leaf water potential than WT, and the decrease of relative accumulativetranspiration rate during the water deficit was alleviated in the transgenic plant. These resultswere summarized that compared to WT, SWPA2 promoter-RWC3 transgenic plant wasendowed with higher water transport of roots and improved water status under water deficit,suggesting aquaporin RWC3 probably play a role during drought avoidance of rice(3) Plant chilling injury was partially caused by water imbalance between water uptakein root and transpiration in leaf. In this study, two rice cultivars (Somewake and Wasetoitsue)were exposed to 7 ± 1°C for up to 24 h and following temperature recovery (28 ± 1°C) for 3h. Throughout the chilling stress, Wasetoitsu showed more serious injury symptoms thanSomewake. By measuring the relative electrical conductivity of rice leaves, Wasetoitsu hadhigher membrane electrolyte leakage than Somewake at the low temperature. These resultsindicated that Wasetoitsue was a less chilling tolerant cultivar compared to Somewake. Thetwo cultivars were in different water status, i.e. Wasetoitsu had lower leaf RWC thanSomewake in the duration of chilling, although both cultivars had decreasing RWC at thesame time RWC. In both cultivars, accumulative transpiration rate and root Lp decreasedobviously during the chilling treatment, but the decrease of Lp was more rapid and acute thanthat of accumulative transpiration rate, suggesting that the water imbalance occurred betweenthe water uptake of root and the water evaporation of aerial parts. These physiologicalobservations prompted us to investigate the expression of water channel proteins. At mRNAlevel, three rice aquaporin genes, RWC1,OsPIP2a and RWC3 showed the similar response tothe chilling treatment in both cultivars, i.e. remained approximate constant within the 9 h withchilling treatment while were decreased after 24 h with chilling treatment. The analysis ofWestern blot with antibody against PIP1 was consistent with the mRNA expression. In bothcultivars, PIP1 protein decreased when the plants were subjected to the chilling treatment for24 h. As we know, this is the first report on aquaporin responses to chilling stress in plantroots. Surprisingly, the temperature recovery for 3 h following chilling treatment resulted incontinued decreases of root PIPs mRNA expression. Aquaporins might be related to ricechilling adaptation but not a key factor of chilling resistance at the early stage of the chillingstress.