Tissue and cell-specific localization of rice aquaporins and their water transport activities

Water transport in plants is greatly dependent on the expression and activity of water transport channels, called aquaporins. Here, we have clarified the tissue- and cell-specific localization of aquaporins in rice plants by immunoblotting and immunocytochemistry using seven isoform-specific aquaporin antibodies. We also examined water transport activities of typical aquaporin family members using a yeast expression system in combination with a stopped-flow spectrophotometry assay. OsPIP1 members, OsPIP2;1, OsTIP1;1 and OsTIP2;2 were expressed in both leaf blades and roots, while OsPIP2;3, OsPIP2;5 and OsTIP2;1 were expressed only in roots. In roots, large amounts of aquaporins accumulated in the region adjacent to the root tip (around 1.5-4 mm from the root tip). In this region, cell-specific localization of the various aquaporin members was observed. OsPIP1 members and OsTIP2;2 accumulated predominantly in the endodermis and the central cylinder, respectively. OsTIP1;1 showed specific localization in the rhizodermis and exodermis. OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated in all root cells, but they showed higher levels of accumulation in endodermis than other cells. In the region at 35 mm from the root tip, where aerenchyma develops, aquaporins accumulated at low levels. In leaf blades, OsPIP1 members and OsPIP2;1 were localized mainly in mesophyll cells. OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;2 expressed in yeast showed high water transport activities. These results suggest that rice aquaporins with various water transport activities may play distinct roles in facilitating water flux and maintaining the water potential in different tissues and cells.     

Characterization of OsPIP2;7, a water channel protein in rice

Aquaporins are water channel proteins that facilitate passage of water and other small neutral molecules across biological membranes. There are usually a large number of members of this family in higher plants, which exhibit various physiological functions and are regulated in a time-specific and particular mode. We have previously shown that a rice gene, OsPIP2;7, was generally up-regulated in roots but down-regulated in shoots at the early stage of chilling stress. Here, OsPIP2;7 was cloned and proved to be an aquaporin with high activity in Xenopus oocytes. OsPIP2;7 was localized mainly in mesophyll cells of leaves. In roots it was detected in the vascular tissues, epidermis cells and exodermis cells at the elongation zone, as well as in the epidermis cells, exodermis cells and root hair at the maturation zone. Yeast cells overexpressing OsPIP2;7 showed a higher survival rate after freeze-thaw stress. Furthermore, OsPIP2;7 enhanced the transpiration rate and tolerance to low temperature when overexpressed in rice. These results indicated that OsPIP2;7 was involved in rapid water transport and maintenance of the water balance in cells, and ultimately improves the tolerance of yeast and rice to low temperature stress.     

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

Plasma membrane intrinsic proteins （PIPs） are a subfamily ofaquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 （OsPIP1-1） or OsPIP2 （OsPIP2-2） in wild-type Arabidopsis, showed enhanced tolerance to salt （100 mM of NaCl） and drought （200 mM ofmannitol）, but not to salt treatment of higher concentration （150 mM of NaCl）. Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation

By means of functional screening using the cadmium (Cd)-sensitiveycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1,from Digitaria ciliaris growing in a former mining area in northernJapan, and have shown that it confers Cd tolerance to the yeastcells, which accumulated almost 2-fold lower Cd levels thancontrol cells. The 521 bp DcCDT1 cDNA contains an open readingframe of 168 bp and encodes a deduced peptide, DcCDT1, thatis 55 amino acid residues in length, of which 15 (27.3%) arecysteine residues. Five DcCDT1 homologs (here termed OsCDT1–OsCDT5)have been identified in rice, and all of them were up-regulatedto varying degrees in the above-ground tissues by CdCl₂ treatment.Localization of green fluorescent protein fusions suggests thatDcCDT1 and OsCDT1 are targeted to both cytoplasmic membranesand cell walls of plant cells. Transgenic Arabidopsis thalianaplants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerantphenotype and, consistent with our yeast data, accumulated loweramounts of Cd when grown on CdCl₂. Collectively, our data suggestthat DcCDT1 and OsCDT1 function to prevent entry of Cd intoyeast and plant cells and thereby enhance their Cd tolerance.     

The chilling injury induced by high root temperature in the leaves of rice seedlings

Root temperature is found to be a very important factor forleaves to alter the response and susceptibility to chillingstress. Severe visible damage was observed in the most activeleaves of seedlings of a japonica rice (Oryza sativa cv. Akitakomachi),e.g. the third leaf at the third-leaf stage, after the treatmentwhere only leaves but not roots were chilled (L/H). On the otherhand, no visible damage was observed after the treatment whereboth leaves and roots were chilled simultaneously (L/L). Thechilling injury induced by L/H, a novel type of chilling injury,required the light either during or after the chilling in orderto develop the visible symptoms such as leaf bleaching and tissuenecrosis. Chlorophyll fluorescence parameters measured aftervarious lengths of chilling treatments showed that significantchanges were induced before the visible injury. The effectivequantum yield and photochemical quenching of PSII dropped dramaticallywithin 24 h in both the presence and absence of a 12 h lightperiod. The maximal quantum yield and non-photochemical quenchingof PSII decreased significantly only in the presence of light.On the other hand, L/H chilling did not affect the functionof PSI, but caused a significant decrease in the electron availabilityfor PSI. These results suggest that the leaf chilling with highroot temperature destroys some component between PSII and PSIwithout the aid of light, which causes the over-reduction ofPSII in the light, and thereby the visible injury is inducedonly in the light.     

Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo.     

Cold Stress-Induced Acclimation in Rice is Mediated by Root-Specific Aquaporins

Cold acclimation process plays a vital role in the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. However, it remains elusive whether a cold acclimation process enhances root water uptake (a component of chilling tolerance) in chilling-sensitive crops such as rice. By analyzing the root hydraulic conductivity under cold stress for a prolonged time, we found that cold stress induced a gradual increase in root osmotic hydraulic conductivity [Lp(r(os))]. Compared with the control treatment (roots and shoots at 25°C), low root temperature (LRT) treatment (roots at 10°C; shoots at 25°C) dramatically reduced Lp(r(os)) within 1 h. However, Lp(r(os)) gradually increased during prolonged LRT treatment and it reached 10-fold higher values at day 5. Moreover, a coordinated up-regulation of root aquaporin gene expression, particularly OsPIP2;5, was observed during LRT treatment. Further, comparison of aquaporin gene expression under root-only chilling (LRT) and whole-plant chilling conditions, and in the roots of intact plants vs. shootless plants, suggests that a shoot to root signal is necessary for inducing the expression of aquaporin genes in the root. Collectively, these results demonstrate that a cold acclimation process for root water uptake functions in rice and is possibly regulated through aquaporins.     

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Fructans are water-soluble fructose oligomers and polymers thatare based on sucrose, and have been implicated in protectingplants against water stress. Rice (Oryza sativa L.) is highlysensitive to chilling temperatures, and is not able to synthesizefructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose1-fructosyltransferase, encoded by wft2, or sucrose:fructan6-fructosyltransferase, encoded by wft1, were introduced intorice plants, and rice transformants that accumulate fructanswere successfully obtained. The mature leaf blades of transgenicrice lines with wft2 or wft1 accumulated 16.2 mg g^–1 FWof oligo- and polysaccharides mainly composed of inulin oligomersof more than DP7, and 3.7 mg g^–1 FW of oligo- and polysaccharides,mainly composed of phlein oligomers of more than DP15, respectively.The transgenic rice seedlings with wft2 accumulated significantlyhigher concentrations of oligo- and polysaccharides than non-transgenicrice seedlings, and exhibited enhanced chilling tolerance. Theoligo- and polysaccharide concentrations of seedlings expressingwft1 were obviously lower than those of lines expressing wft2,and no correlation between oligo- and polysaccharide concentrationsand chilling tolerance was detected in wft1-expressing ricelines. The results suggest that transgenic rice lines expressingwheat-derived fructosyltransferase genes accumulated large amountsof fructans in mature leaf blades and exhibited enhanced chillingtolerance at the seedling stage. This is the first report owingthat fructan accumulation enhanced tolerance to non-freezinglow temperatures. Key words: Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant     

Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulatephotoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 andHd3a were identified as orthologs of GI, CO and FT, respectively,and are also important regulators of flowering. Although GIhas roles in both flowering and the circadian clock, our understandingof its biochemical functions is still limited. In this study,we purified novel OsGI-interacting proteins by using the tandemaffinity purification (TAP) method. The TAP method has beenused effectively in a number of model species to isolate proteinsthat interact with proteins of interest. However, in plants,the TAP method has been used in only a few studies, and no novelproteins have previously been isolated by this method. We generatedtransgenic rice plants and cell cultures expressing a TAP-taggedversion of OsGI. After a two-step purification procedure, theinteracting proteins were analyzed by mass spectrometry. Sevenproteins, including dynamin, were identified as OsGI-interactingproteins. The interaction of OsGI with dynamin was verifiedby co-immunoprecipitation using a myc-tagged version of OsGI.Moreover, an analysis of Arabidopsis dynamin mutants indicatedthat although the flowering times of the mutants were not differentfrom those of wild-type plants, an aerial rosette phenotypewas observed in the mutants. We also found that OsGI is presentin both the nucleus and the cytosol by Western blot analysisand by transient assays. These results indicate that the TAPmethod is effective for the isolation of novel proteins thatinteract with target proteins in plants.     

Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina

Selaginella tamariscina, one of the most primitive vascularplants, can remain alive in a desiccated state and resurrectwhen water becomes available. To evaluate the nature of desiccationtolerance in this plant, we compared the composition of solublesugars and saturation ratios of phospholipids (PLs) betweenhydrated and desiccated tissues of S. tamariscina using gaschromatography. In this study, differences in gene expressionand ABA contents were also analyzed during dehydration. Theresults revealed that trehalose (at >130 mg g^–1 DW)was the major soluble sugar, and low saturated fatty acid contentin PLs (0.31) was maintained in both hydrated and desiccatedtissues. In addition, the ABA content of S. tamariscina increased3-fold, and genes involved in ABA signaling and cellular protectionwere up-regulated while photosystem-related genes were down-regulatedduring dehydration. The biochemical and molecular findings suggestthat both constitutive and inducible protective molecules contributeto desiccation tolerance of S. tamariscina.     

Identification of maize silicon influx transporters

Maize (Zea mays L.) shows a high accumulation of silicon (Si),but transporters involved in the uptake and distribution havenot been identified. In the present study, we isolated two genes(ZmLsi1 and ZmLsi6), which are homologous to rice influx Sitransporter OsLsi1. Heterologous expression in Xenopus laevisoocytes showed that both ZmLsi1 and ZmLsi6 are permeable tosilicic acid. ZmLsi1 was mainly expressed in the roots. By contrast,ZmLsi6 was expressed more in the leaf sheaths and blades. Differentfrom OsLsi1, the expression level of both ZmLsi1 and ZmLsi6was unaffected by Si supply. Immunostaining showed that ZmLsi1was localized on the plasma membrane of the distal side of rootepidermal and hypodermal cells in the seminal and crown roots,and also in cortex cells in lateral roots. In the shoots, ZmLsi6was found in the xylem parenchyma cells that are adjacent tothe vessels in both leaf sheaths and leaf blades. ZmLsi6 inthe leaf sheaths and blades also exhibited polar localizationon the side facing towards the vessel. Taken together, it canbe concluded that ZmLsi1 is an influx transporter of Si, whichis responsible for the transport of Si from the external solutionto the root cells and that ZmLsi6 mainly functions as a Si transporterfor xylem unloading.