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Aquaporins in developing rice grains ab
a
a
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Hidehiro Hayashi , Junko Ishikawa-Sakurai , Mari Murai-Hatano , Arifa Ahamed & bc
Matsuo Uemura a
Agro-Production Technologies and Management Research Division, NARO Tohoku Agricultural Research Center, Morioka, Japan b
United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan
c
Faculty of Agriculture, Cryobiofrontier Research Center, Iwate University, Morioka, Japan Published online: 17 Apr 2015.
Click for updates To cite this article: Hidehiro Hayashi, Junko Ishikawa-Sakurai, Mari Murai-Hatano, Arifa Ahamed & Matsuo Uemura (2015): Aquaporins in developing rice grains, Bioscience, Biotechnology, and Biochemistry To link to this article: http://dx.doi.org/10.1080/09168451.2015.1032882
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Bioscience, Biotechnology, and Biochemistry, 2015
Aquaporins in developing rice grains Hidehiro Hayashi1,2,*, Junko Ishikawa-Sakurai1,†, Mari Murai-Hatano1, Arifa Ahamed3 and Matsuo Uemura2,3 1
Agro-Production Technologies and Management Research Division, NARO Tohoku Agricultural Research Center, Morioka, Japan; 2United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan; 3Faculty of Agriculture, Cryobiofrontier Research Center, Iwate University, Morioka, Japan Received December 15, 2014; accepted March 12, 2015
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http://dx.doi.org/10.1080/09168451.2015.1032882
During rice grain filling, grain moisture content and weight show dynamic changes. We focused on the expression of all 33 rice aquaporins in developing grains. Only two aquaporin genes, OsPIP2;1 and OsTIP3;1, were highly expressed in the period 10–25 days after heading (DAH). High-temperature treatment from 7 to 21 DAH abolished the dynamic up-regulation of OsPIP2;1 in the period 15–20 DAH, whereas OsTIP3;1 expression was not affected. Immunohistochemical analysis revealed that OsPIP2;1 was present in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer. OsTIP3;1 was present in the aleurone layer and starchy endosperm. Water transport activity of recombinant OsTIP3;1 was low, in contrast to the high activity of recombinant OsPIP2;1 we reported previously. Our data suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains. Key words:
aleurone layer; aquaporin; developing grain; Oryza sativa; phloem
Cereals produce grains for reproduction, and people use these grains for food. Grains are the edible parts of the three major food crops, wheat, rice, and maize. Rice (Oryza sativa) is the most important staple crop in monsoon Asia that supports the world’s largest population. Today, rice is grown not only in Asia, but also in Europe and America. During rice grain filling, grain moisture content and weight change dynamically as a result of the transport of large amounts of assimilates; this transport is linked to water movement in developing grains. In recent years, high temperature during rice grain filling is causing a decrease in rice grain weight and quality. The assimilate transporting and water movement may be involved in these process. According to the pressure flow hypothesis,1) the dorsal
vascular bundles in developing grains (a sink) maintain a low turgor pressure, which may cause phloem flows from source organs to allow assimilate accumulation in the starchy endosperm. It has been proposed that xylem vessels in dorsal vascular bundles function as drain pipes to maintain low turgor pressure in the phloem.2–4) Furthermore, no transpiration stream from the roots has been observed in the xylem in dorsal vascular bundles,4,5) and the transpiration rate from rice grains is related to grain dry matter increase.6) However, the mechanism of efficient control of water flow from the phloem to the xylem vessels in the dorsal vascular bundles is unclear. The pathways for transporting assimilates and water into the developing grain were proposed.3,7–9) After aleurone layer differentiation (6–7 days after anthesis),10) these pathways have to through the aleurone layer.11) Therefore, the aleurone layer may play important roles in water and solute transport. All of these processes might be mediated by water channels (aquaporins) and solute channels, but the relation between grain development and aquaporins remains largely unexplored. The rice genome encodes 33 aquaporins12): plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), and small and basic intrinsic proteins. Previous studies have characterized gene expression in response to environmental factors, and the abundance, localization, and water transport activity of these aquaporins.12–18) These studies suggested that individual aquaporins play specific roles in plant water homeostasis in response to various environmental stresses19); they also transport small uncharged solutes such as boron,20,21) silica,22) CO2,23–26) arsenic trioxide,27,28) and glycerol.29) In comparison with extensive studies of aquaporins in roots30) and leaves,31) only limited information is available on aquaporins in reproductive organs. TIP3 is expressed in seeds, and the protein is located in the membranes of protein storage vacuoles
*Corresponding author. Email: [email protected] † Present address: Rice Research Division, NARO Institute of Crop Science, Tsukuba 305-8518, Japan. Abbreviations: CT, control temperature treatment; DAF, days after flowering; DAH, days after heading; HT, high temperature treatment; NIP, nodulin 26-like intrinsic protein; Pf, osmotic water permeability coefficient; PIP, plasma membrane intrinsic protein; PSV, protein storage vacuole; TIP, tonoplast intrinsic protein. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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32,33)
(PSV) of Glycine max. In Phaseolus vulgaris, water permeability mediated by TIP3 is modulated by its phosphorylation.34) In rice, OsTIP3;1 and OsTIP3;2 are specifically expressed in developing grains.35,36) OsTIP1;1 is located in PSV membranes only, whereas OsTIP3;1 is located in the PSV and the aleurone grain membranes in developing grains 10 days after flowering (DAF).35) Li et al. reported that OsTIP3;2 has a glycerol transport activity but not water transport activity in a Xenopus oocyte expression system.36) However, little is known about the roles of TIP3 and other aquaporins in developing rice grains. The main purpose of this study was to detect aquaporins involved in rice grain development. Among the 33 rice aquaporins, characteristic patterns of expression of several genes were detected. OsPIP2;1 and OsTIP3;1 were highly expressed in developing grains in the mid-grain filling stage (10–25 days after heading [DAH]). Our data also suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains.
Materials and methods Plant materials. Rice (O. sativa L. cv. Akitakomachi) seeds were germinated in the dark for 3 days at room temperature. Thereafter, the seedlings were grown in a growth chamber (PGW36; Conviron, Manitoba, Canada) under a 12 h light (6:30–18:30)/12 h dark (18:30–6:30) photoperiod (at a photosynthetic photon flux density of 400 μmol s−1 m−2) at 25/20 °C and a relative humidity of 75%. Plants were grown in 1/5000-a Wagner pots with flooded soil containing 0.9 g nitrogen, 0.39 g phosphorus, 0.75 g potassium, 0.18 g magnesium, 0.06 g iron, 0.04 g manganese, and 0.22 g calcium. During grain filling, 1 tiller per plant was taken from at least three plants, and the developing grains were collected. To obtain uniform samples, the 3rd to the 5th grains from the top on the 1st to the 3rd primary branches were selected. In the present study, the above grains bloomed 1 or 2 DAH because they were located near the top. RNA extraction and quantitative real-time PCR. Developing grains were removed from the palea and lemma. The grains were immediately frozen in liquid nitrogen and ground with a mortar and pestle. Total RNA was extracted using Fruit-mate (Takara Bio, Otsu, Shiga, Japan), RNAiso Plus (Takara Bio), RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), and RNase-free DNase set (Qiagen) according to the manufacturer’s instructions. Frozen powdered tissue (50 mg) was suspended in 1 ml of Fruit-mate and centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was mixed with an equal volume of RNAiso Plus, incubated at room temperature for 5 min, mixed with 0.2 volumes of chloroform, and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous top layer was mixed with 0.5 volumes of ethanol, transferred into an RNeasy spin column, and centrifuged at 8000 × g for 15 s at room temperature; the flow-through was discarded. The subsequent steps for total RNA extraction were conducted using an RNeasy Plant Mini Kit and RNase-free DNase set. First-strand cDNA was
synthesized from 1.5 μg of total RNA using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with diluted cDNA (1:50) using a StepOne real-time PCR system and Fast SYBR Green system (both from Applied Biosystems) with the primer sets listed in Supplementary Table S1 and 18S rRNA as an internal control. Absolute mRNA copy numbers per pg of total RNA were calculated using standard plasmids carrying each aquaporin cDNA as reported by Sakurai-Ishikawa et al.16) Immunoblotting. Developing grains collected with the palea and lemma were immediately frozen in liquid nitrogen and ground with a mortar and pestle. Frozen powder was mixed with 1% (w/v) SDS, 2 mM EDTA, 20 mM Tris–HCl (pH 7.6), centrifuged at 13,000 × g for 10 min, and the supernatants were used as total protein for immunoblotting. SDS-PAGE was performed in 12.5% polyacrylamide gels, and proteins were transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA) in a semi-dry blotting apparatus for 40 min at 15 V. The membrane was blocked with 4% (w/v) skim milk in Tris-buffered saline and incubated with aquaporin antibody (1:1000 dilution). Anti-OsPIP2;1 and anti-OsTIP3;1 antibodies were raised against peptides MGKDEVMESGGAAGEFAAKDY14) and MSTAAARPGRRFTVGRS, respectively. Signals were visualized using horseradish peroxidase-linked Protein-A (1:2000 dilution) (GE Healthcare BioSciences, Piscataway, NJ, USA) and ECL detection reagents (GE Healthcare BioSciences).
High-temperature treatment. Temperature treatment was conducted during 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH in a growth chamber at 25/ 20 °C (control temperature treatment [CT]) or 32/27 °C (High-temperature treatment [HT]) day/night cycles. During grain filling, developing grains were collected and removed from the palea and lemma, and the fresh weight (FW) of each grain was measured immediately. Dry weight (DW) of developing grains was measured after oven-drying for more than 72 h at 80 °C. The relative moisture content in grains was evaluated from the ratio between fresh and DWs as (FW − DW)/ FW × 100. The moisture content of developing grains was evaluated from the difference between fresh and DWs (FW − DW). Immunohistochemical fluorescence staining. The 12-DAH grains were fixed in a solution containing 4% (w/v) paraformaldehyde, 0.08% glutaraldehyde, 5.0% acetic acid, and 50% ethanol. The fixed grains were first dehydrated through a graded ethanol series, then cleared with xylene, embedded in paraffin blocks, and sectioned vertically into 30-μm slices on a Leica RM2125RT microtome (Leica Microsystems GmbH, Nussloch, Germany). The 22-DAH or fully mature grains were cut vertically into halves with a razor blade, fixed in 4% (w/v) paraformaldehyde and 0.08%
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glutaraldehyde in 100 mM sodium phosphate buffer (pH 7.2), and dehydrated through a graded ethanol series. Grain sections and halves were blocked with 1% (w/v) bovine serum albumin in phosphate-buffered saline and 0.2% (w/v) Tween-20 and incubated with anti-aquaporin antibodies or pre-immune serum (1:100 dilution). After washing, Alexa Fluor 555 goat anti-rabbit IgG (1:200 dilution) (Invitrogen, Carlsbad, CA, USA) was used for visualizing aquaporins. Fluorescence was observed with an IX81 microscope equipped with a U-LH75XEAPO xenon burner and U-MWIG3 mirror unit (Olympus, Tokyo, Japan). Expression of rice aquaporins in yeast and determination of membrane osmotic water permeability. Rice aquaporin cDNA fragments were inserted into a yeast expression vector37,38) and expressed in Saccharomyces cerevisiae strain BJ5458 as described previously.12) Crude yeast membrane fractions were prepared according to the method reported previously.14) Membrane osmotic water permeability was measured by the stopped-flow light scattering method as described previously (stopped-flow spectrophotometer model SX18MV; Applied Photo Physics, Surrey, UK).14) Yeast membrane vesicles in 200 mM mannitol solution were quickly mixed with an equal volume of 400 mM mannitol solution. The mannitol solution contained 200 mM or 400 mM mannitol, 90 mM KCl, 1 mM EDTA, 1 mM EGTA, and 20 mM Tris–HCl, pH 7.2. Vesicle shrinking was monitored at 10 °C as an increase in scattered light intensity. Water transport activity was evaluated as membrane osmotic water permeability. Osmotic water permeability coefficient (Pf) was calculated from the initial rate constants (k), which were calculated from exponential fitting: Pf = (V0 × k)/(S × VW × Cout). The initial vesicle volume (V0) and the initial vesicle surface area (S) in 200 mM mannitol were calculated to be 7.63 × 10−3 μm3 and 1.87 × 10−1 μm2, respectively, according to the yeast membrane vesicle diameter in 450 mM mannitol.38) The partial molar volume of water (VW) was set as 18 cm3 mol−1. The initial concentrations of total solute outside the vesicle (Cout) were calculated from the concentration of 400 mM mannitol solution. The relative level of each aquaporin protein was calculated from signal intensity upon blot staining with anti-myc antibody using CS Analyzer ver 3.0 (ATTO, Tokyo, Japan).
Fig. 1. Dynamic changes in aquaporin expression levels and grain morphology during the grain filling period. Notes: The areas between the lines show the expression levels of each gene. Values are means of three independent growth experiments. To collect the grains, 1 tiller per plant was taken from at least three plants every 5 days. The 3rd to the 5th grains from the top on the 1st to the 3rd primary branches were selected. The transcript levels were normalized to the amounts of 18S rRNA. Aquaporins with 0.25 mRNA copies per pg total RNA) at the flowering stage (0 DAH, whole pistil). In the mid-grain filling stage, only several aquaporin transcripts, including OsPIP2;1, OsTIP2;2, and OsTIP3;1 were abundant (Fig. 1). Among them, OsPIP2;1 and OsTIP3;1 transcripts were predominant and peaked at 15 and 20 DAH, respectively (Fig. 1, see also Fig. 4). These two aquaporin
Fig. 2. Dynamic changes in the protein levels of OsPIP2;1 (A) and OsTIP3;1 (B) in developing grains. Note: Total protein (5 µg per lane) was subjected to immunoblotting with anti-aquaporin antibodies.
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(Fig. 2). Thus, the changes in the abundance of these two aquaporins were similar at the protein level.
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Effect of high temperature on dry matter accumulation, moisture content, and aquaporin expression in developing grains To evaluate the effect of high air temperature on dry matter accumulation and relative moisture content during grain filling, we measured fresh and DWs of the grains at different time points. Under CT, both parameters increased during the period from 5 to 20 DAH:
FW from 3.3 to 27.6 mg, and DW from 0.6 to 19.6 mg (Fig. 3(A)). A similar increase was observed under HT: FW increased from 3.3 to 23.3 mg and DW increased from 0.6 to 15.1 mg (Fig. 3(A)). During the same period, the relative moisture content decreased rapidly in both treatments and reached 28.9% in CT, whereas in HT, it remained 6.4 percentage points higher at 20 DAH (Fig. 3(B)). From 20 DAH, the relative moisture content stabilized in both treatments. At 45 DAH, the relative moisture content was 9.7 percentage points higher in HT than in CT (Fig. 3(B)). From 5 to 10 DAH, a rapid increase was observed in the moisture content per grain (Fig. 3(C)), indicating high water influx in this stage. In the mid-grain filling stage, the moisture content per grain decreased, indicating water efflux from the developing grain in this stage (Fig. 3(C)). The moisture content per grain was higher in HT than in CT from 30 DAH (Fig. 3(C)). We examined the effect of high air temperature on the gene expression levels of the two major aquaporins of developing grains. HT inhibited the increase in OsPIP2;1 expression during the period from 10 to 20 DAH (Fig. 4(A)) but did not affect that of OsTIP3;1 (Fig. 4(B)). Immunohistochemistry of aquaporins in rice grains In the 12-DAH grain sections, OsPIP2;1 was detected in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer (Fig. 5(A) and (D)). OsTIP3;1
Fig. 3. Effect of high temperature on FW and DW (A), the relative moisture content (B), and the moisture content per grain (C) in developing grains. Notes: Temperature treatment was conducted for 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH (time between the two broken lines) in a growth chamber at 25/20 °C (CT) or 32/27 °C (HT) day/ night cycles. Values are means ± SE of three independent growth experiments. Statistically significant differences between CT and HT at each DAH (Student’s t-test) are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4. Effect of high temperature on the expression of OsPIP2;1 (A) and OsTIP3;1 (B) in developing grains. Notes: Treatment was conducted for 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH (time between the two broken lines) in a growth chamber at 25/20 °C (CT) or 32/27 °C (HT) day/night cycles. Values are means ± SE of three independent growth experiments. The transcript levels were normalized to the amounts of 18S rRNA. **p < 0.01, statistically significant differences between CT and HT at each DAH (Student’s t-test).
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Fig. 5. Localization of OsPIP2;1 and OsTIP3;1 in rice grains. Notes: The 12-DAH grain sections, and halves of 22-DAH and fully mature grains were labeled with respective anti-aquaporin antibodies and visualized with secondary antibody conjugated with Alexa Fluor 555 (red). AL, aleurone layer; DV, dorsal vascular bundles; NE, nucellar epidermis; NP, nucellar projection; PH, phloem; SE, starchy endosperm; XV, xylem vessels. Scale bars, 100 μm.
was detected in the aleurone layer and starchy endosperm, but not in the nucellar projection, nucellar epidermis, and dorsal vascular bundles (Fig. 5(B) and (E)). In the 22-DAH grains, OsPIP2;1 was detected mainly in the starchy endosperm (Fig. 5(F)), whereas OsTIP3;1 was found mainly in the aleurone layer (Fig. 5(G)). In the fully mature grains (from 45 DAH), OsTIP3;1 localization was similar to that in the 22DAH grains (Fig. 5(J)), whereas OsPIP2;1 staining became weak and difficult to detect (Fig. 5(I)).
Water transport activity of OsTIP3;1 We compared water transport activity of OsTIP3;1 and used OsPIP2;5 which has high water transport activity12,14) as a positive control and the vector as a negative control. The membrane fractions prepared from the yeast cells expressing rice aquaporins showed immunostained bands close to the expected molecular sizes of aquaporin monomers and dimers (Fig. 6(A)). The water transport activity of OsPIP2;5 was approx. 2.5 times that of the vector control. The water transport activity of OsTIP3;1 was similar to that of the vector control (Fig. 6(B)).
Discussion In this study, we investigated the expression of all 33 rice aquaporin genes in developing grains and found that only OsPIP2;1 and OsTIP3;1 transcripts were highly abundant in the mid-grain filling stage. Our data suggest that these two aquaporins might play different roles. OsPIP2;1 in grain filling Our study is the first to address the importance of OsPIP2;1 in grain filling. OsPIP2;1 was expressed
Fig. 6. The osmotic water permeability of yeast membrane vesicles. Notes: Crude membrane fractions prepared from yeasts expressing aquaporin constructs were subjected to immunoblotting with anti-myc antibody (A); 1 µg total protein was applied to each lane. Osmotic water permeability coefficient (Pf) of crude membrane fractions from cells producing recombinant OsPIP2;5 or OsTIP3;1, or cells carrying myc-pKT10 as vector control (B). Pf was corrected for the expression level of each aquaporin protein. The values are means ± SE of three individual replicates A–C (A). *p < 0.05, statistically significant difference from myc-pKT10 (Student’s t-test).
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Fig. 7. The proximal promoter region and 5′-UTR of OsTIP3;1. Note: ABRE-like and AW-box motifs are underlined. TSS, transcription start site. ATG, translation initiation codon.
abundantly in many organs, including developing grains (Fig. 1), leaves, and roots.12,18) From 5 to 10 DAH, OsPIP2;1 was expressed abundantly in developing grains together with several other aquaporins (Fig. 1). The water efflux rate was the highest during the period from 10 to 20 DAH (Fig. 3(C)), and at that time, OsPIP2;1 and OsTIP3;1 were most abundant (Figs. 1 and 4). Thus, we hypothesized that OsPIP2;1, OsTIP3;1, or both are important for net water efflux, dry matter accumulation, or both in developing grains after the mid-grain filling stage. Using the stopped-flow light scattering method, we have previously found that water transport activity of OsPIP2;1 was high.14) OsPIP2;1 was detected in many grain tissues (Fig. 5(A), (D), (F) and (I)), indicating its involvement in the cell-to-cell water transport pathway. We found that HT abolished the dynamic up-regulation of OsPIP2;1 during the mid-grain filling stage (Fig. 4(A)). Simultaneously, HT suppressed dry matter accumulation (Fig. 3(A)). OsPIP2;1 was located in the dorsal vascular bundles where the phloem and xylem vessels are located (Fig. 5(A) and (D)). OsPIP2;1 may enhance the phloem flow by discharging water from the phloem to the xylem. We propose that abolishing the dynamic up-regulation of OsPIP2;1 during the mid-grain filling stage may suppress dry matter accumulation under HT. High temperature during grain filling decreases rice grain weight and quality.39) Various mechanisms, such as changes in gene expression40,41) and nitrogen availability to rice plants,42) have been suggested to explain these effects. A decrease in water permeability due to the decreased OsPIP2;1 expression may be one of these mechanisms. Ishimaru et al. found that loose packing of grain amyloplasts leads to a higher moisture content under high temperature.43) Their observation is supported by our data that the relative water content in grains was higher in HT than in CT (Fig. 3(B)). OsTIP3;1 in grain filling The OsTIP3;1 transcript level from 15 to 25 DAH was not affected by HT (Fig. 4(B)), although dry matter accumulation during this period was reduced by HT (Fig. 3(A)). OsTIP3;1 had low water transport activity (Fig. 6(B)). These results suggest that OsTIP3;1 does not contribute considerably to the water flux, and HT induced decrease in the dry matter accumulation in the grain. Using immunohistochemical analysis, we detected OsTIP3;1 mainly in the aleurone layer in the 12-DAH, 22-DAH, and fully mature grains (Fig. 5(B), (E), (G) and (J)), whereas OsPIP2;1 was not observed
in the aleurone layer of the 12-DAH grains (Fig. 5(A) and (D)). These results suggest that the aleurone layer has low water permeability. For efficient assimilate transport, prompt absorption of water from phloem sap to xylem vessels may be needed.2–4) We believe that, to control water flow direction, the 12-DAH grains maintain high water permeability in the dorsal vascular bundles and low water permeability in the aleurone layer. Among all rice aquaporin genes, only OsTIP3;1 has 2 ABRE44)-like sequences located 200 bp upstream of the transcription start site in the proximal promoter region (Fig. 7); this indicates that a temporal increase in the OsTIP3;1 transcript level during grain filling might be mediated by ABA signaling. The aleurone layer accumulates triacylglycerols in oil bodies and phytin in the aleurone grains45) during the same stage of grain filling as when OsTIP3;1 is expressed. OsTIP3;1 has AW-boxes46) in the proximal promoter region (Fig. 7). The transcription factor WRINKLED1, required for triacylglycerol accumulation, binds to AWboxes. Thus, OsTIP3;1 may be involved in some of these metabolic pathways. In the present study, we did not examine the role of TIP3 phosphorylation in the regulation of water transport activity.34) We cannot rule out the possibility that OsTIP3;1 has water transport activity on another experimental condition or in planta. Further studies are needed to reveal OsTIP3;1 functions.
Supplementary material The supplementary material for this paper is available at http://dx.doi.org/10.1080/09168451.2015. 1032882.
Acknowledgments We are grateful to Prof. Masayoshi Maeshima for providing aquaporin antibodies and for valuable discussions throughout this work. We are grateful to Prof. Maki Katsuhara for valuable suggestions throughout this work.
Disclosure statement No potential conflict of interest was reported by the authors.
Aquaporins in developing rice grains
Funding This work was partially supported by the Bio-oriented Technology Research Advancement Institute (Brain) of Japan [Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) to M.M.]; the NARO Gender Equality Program.
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[19] Maurel C, Javot H, Lauvergeat V, Gerbeau P, Tournaire C, Santoni V, Heyes J. Molecular physiology of aquaporins in plants. Int. Rev. Cytol. 2002;215:105–148. [20] Takano J, Wada M, Ludewig U, Schaaf G, von, Wiren N, Fujiwara T. The arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. Plant Cell. 2006;18:1498–1509. [21] Tanaka M, Fujiwara T. Physiological roles and transport mechanisms of boron: perspectives from plants. Pflugers Arch. 2008;456:671–677. [22] Ma JF, Tamai K, Yamaji N, Mitani N, Konishi S, Katsuhara M, Ishiguro M, Murata Y, Yano Y. A silicon transporter in rice. Nature. 2006;440:668–691. [23] Uehlein N, Lovisolo C, Siefritz F, Kaldenhoff R. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature. 2003;425:734–737. [24] Hanba YT, Shibasaka M, Hayashi Y, Hayakawa T, Kasamo K, Terashima I, Katsuhara M. Overexpression of the barley aquaporin HvPIP2;1 increases internal CO2 conductance and CO2 assimilation in the leaves of transgenic rice plants. Plant Cell Physiol. 2004;45:521–529. [25] Flexas J, Ribas-Carbó M, Hanson DT, Bota J, Otto B, Cifre J, McDowell N, Medrano H, Kaldenhoff R. Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. Plant J. 2006;48:427–439. [26] Mori IC, Rhee J, Shibasaka M, Sasano S, Kaneko T, Horie T, Katsuhara M. CO2 transport by PIP2 aquaporins of barley. Plant Cell Physiol. 2014;55:251–257. [27] Ma JF, Yamaji N, Mitani N, Xu X, Su Y, McGrath SP, Zhao FJ. Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc. Natl. Acad. Sci. 2008;105:9931–9935. [28] Kamiya T, Tanaka M, Mitani N, Ma JF, Maeshima M, Fujiwara T. NIP1;1, an aquaporin homolog, determines the arsenite sensitivity of Arabidopsis thaliana. J. Biol. Chem. 2009;284:2114– 2120. [29] Gerbeau P, Guclu J, Ripoche P, Maurel C. Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes. Plant J. 1999;18:577–587. [30] Vandeleur R, Niemietz C, Tilbrook J, Tyerman SD. Roles of aquaporins in root responses to irrigation. Plant Soil. 2005;274:141–161. [31] Sack L, Holbrook NM. Leaf hydraulics. Annu. Rev. Plant Biol. 2006;57:361–381. [32] Johnson KD, Herman EM, Chrispeels MJ. An abundant, highly conserved tonoplast protein in seeds. Plant Physiol. 1989;91:1006–1013. [33] Melroy DL, Herman EM. TIP, an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal. Planta. 1991;184:113–122. [34] Maurel C, Kado RT, Guern J, Chrispeels MJ. Phosphorylation regulates the water channel activity of the seed-specific aquaporin α-TIP. EMBO J. 1995;14:3028–3035. [35] Takahashi H, Rai M, Kitagawa T, Morita S, Masumura T, Tanaka K. Differential localization of tonoplast intrinsic proteins on the membrane of protein body type II and aleurone grain in rice seeds. Biosci. Biotechnol. Biochem. 2004;68:1728–1736. [36] Li GW, Peng YH, Yu X, Zhang MH, Cai WM, Sun WN, Su WA. Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice. J. Plant Physiol. 2008;165:1879–1888. [37] Tanaka K, Nakafuku M, Tamanoi F, Kaziro Y, Matsumoto K, Toh-e A. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol. Cell. Biol. 1990;10:4303–4313. [38] Suga S, Maeshima M. Water channel activity of radish plasma membrane aquaporins heterologously expressed in yeast and their modification by site-directed mutagenesis. Plant Cell Physiol. 2004;45:823–830. [39] Peng S, Huang J, Sheehy JE, Laza RC, Visperas RM, Zhong X, Centeno GS, Khush GS, Cassman KG. Rice yields decline with higher night temperature from global warming. Proc. Natl Acad. Sci. USA. 2004;101:9971–9975.
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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Aquaporins in developing rice grains ab
a
a
c
Hidehiro Hayashi , Junko Ishikawa-Sakurai , Mari Murai-Hatano , Arifa Ahamed & bc
Matsuo Uemura a
Agro-Production Technologies and Management Research Division, NARO Tohoku Agricultural Research Center, Morioka, Japan b
United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan
c
Faculty of Agriculture, Cryobiofrontier Research Center, Iwate University, Morioka, Japan Published online: 17 Apr 2015.
Click for updates To cite this article: Hidehiro Hayashi, Junko Ishikawa-Sakurai, Mari Murai-Hatano, Arifa Ahamed & Matsuo Uemura (2015): Aquaporins in developing rice grains, Bioscience, Biotechnology, and Biochemistry To link to this article: http://dx.doi.org/10.1080/09168451.2015.1032882
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Bioscience, Biotechnology, and Biochemistry, 2015
Aquaporins in developing rice grains Hidehiro Hayashi1,2,*, Junko Ishikawa-Sakurai1,†, Mari Murai-Hatano1, Arifa Ahamed3 and Matsuo Uemura2,3 1
Agro-Production Technologies and Management Research Division, NARO Tohoku Agricultural Research Center, Morioka, Japan; 2United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan; 3Faculty of Agriculture, Cryobiofrontier Research Center, Iwate University, Morioka, Japan Received December 15, 2014; accepted March 12, 2015
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http://dx.doi.org/10.1080/09168451.2015.1032882
During rice grain filling, grain moisture content and weight show dynamic changes. We focused on the expression of all 33 rice aquaporins in developing grains. Only two aquaporin genes, OsPIP2;1 and OsTIP3;1, were highly expressed in the period 10–25 days after heading (DAH). High-temperature treatment from 7 to 21 DAH abolished the dynamic up-regulation of OsPIP2;1 in the period 15–20 DAH, whereas OsTIP3;1 expression was not affected. Immunohistochemical analysis revealed that OsPIP2;1 was present in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer. OsTIP3;1 was present in the aleurone layer and starchy endosperm. Water transport activity of recombinant OsTIP3;1 was low, in contrast to the high activity of recombinant OsPIP2;1 we reported previously. Our data suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains. Key words:
aleurone layer; aquaporin; developing grain; Oryza sativa; phloem
Cereals produce grains for reproduction, and people use these grains for food. Grains are the edible parts of the three major food crops, wheat, rice, and maize. Rice (Oryza sativa) is the most important staple crop in monsoon Asia that supports the world’s largest population. Today, rice is grown not only in Asia, but also in Europe and America. During rice grain filling, grain moisture content and weight change dynamically as a result of the transport of large amounts of assimilates; this transport is linked to water movement in developing grains. In recent years, high temperature during rice grain filling is causing a decrease in rice grain weight and quality. The assimilate transporting and water movement may be involved in these process. According to the pressure flow hypothesis,1) the dorsal
vascular bundles in developing grains (a sink) maintain a low turgor pressure, which may cause phloem flows from source organs to allow assimilate accumulation in the starchy endosperm. It has been proposed that xylem vessels in dorsal vascular bundles function as drain pipes to maintain low turgor pressure in the phloem.2–4) Furthermore, no transpiration stream from the roots has been observed in the xylem in dorsal vascular bundles,4,5) and the transpiration rate from rice grains is related to grain dry matter increase.6) However, the mechanism of efficient control of water flow from the phloem to the xylem vessels in the dorsal vascular bundles is unclear. The pathways for transporting assimilates and water into the developing grain were proposed.3,7–9) After aleurone layer differentiation (6–7 days after anthesis),10) these pathways have to through the aleurone layer.11) Therefore, the aleurone layer may play important roles in water and solute transport. All of these processes might be mediated by water channels (aquaporins) and solute channels, but the relation between grain development and aquaporins remains largely unexplored. The rice genome encodes 33 aquaporins12): plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), and small and basic intrinsic proteins. Previous studies have characterized gene expression in response to environmental factors, and the abundance, localization, and water transport activity of these aquaporins.12–18) These studies suggested that individual aquaporins play specific roles in plant water homeostasis in response to various environmental stresses19); they also transport small uncharged solutes such as boron,20,21) silica,22) CO2,23–26) arsenic trioxide,27,28) and glycerol.29) In comparison with extensive studies of aquaporins in roots30) and leaves,31) only limited information is available on aquaporins in reproductive organs. TIP3 is expressed in seeds, and the protein is located in the membranes of protein storage vacuoles
*Corresponding author. Email: [email protected] † Present address: Rice Research Division, NARO Institute of Crop Science, Tsukuba 305-8518, Japan. Abbreviations: CT, control temperature treatment; DAF, days after flowering; DAH, days after heading; HT, high temperature treatment; NIP, nodulin 26-like intrinsic protein; Pf, osmotic water permeability coefficient; PIP, plasma membrane intrinsic protein; PSV, protein storage vacuole; TIP, tonoplast intrinsic protein. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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32,33)
(PSV) of Glycine max. In Phaseolus vulgaris, water permeability mediated by TIP3 is modulated by its phosphorylation.34) In rice, OsTIP3;1 and OsTIP3;2 are specifically expressed in developing grains.35,36) OsTIP1;1 is located in PSV membranes only, whereas OsTIP3;1 is located in the PSV and the aleurone grain membranes in developing grains 10 days after flowering (DAF).35) Li et al. reported that OsTIP3;2 has a glycerol transport activity but not water transport activity in a Xenopus oocyte expression system.36) However, little is known about the roles of TIP3 and other aquaporins in developing rice grains. The main purpose of this study was to detect aquaporins involved in rice grain development. Among the 33 rice aquaporins, characteristic patterns of expression of several genes were detected. OsPIP2;1 and OsTIP3;1 were highly expressed in developing grains in the mid-grain filling stage (10–25 days after heading [DAH]). Our data also suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains.
Materials and methods Plant materials. Rice (O. sativa L. cv. Akitakomachi) seeds were germinated in the dark for 3 days at room temperature. Thereafter, the seedlings were grown in a growth chamber (PGW36; Conviron, Manitoba, Canada) under a 12 h light (6:30–18:30)/12 h dark (18:30–6:30) photoperiod (at a photosynthetic photon flux density of 400 μmol s−1 m−2) at 25/20 °C and a relative humidity of 75%. Plants were grown in 1/5000-a Wagner pots with flooded soil containing 0.9 g nitrogen, 0.39 g phosphorus, 0.75 g potassium, 0.18 g magnesium, 0.06 g iron, 0.04 g manganese, and 0.22 g calcium. During grain filling, 1 tiller per plant was taken from at least three plants, and the developing grains were collected. To obtain uniform samples, the 3rd to the 5th grains from the top on the 1st to the 3rd primary branches were selected. In the present study, the above grains bloomed 1 or 2 DAH because they were located near the top. RNA extraction and quantitative real-time PCR. Developing grains were removed from the palea and lemma. The grains were immediately frozen in liquid nitrogen and ground with a mortar and pestle. Total RNA was extracted using Fruit-mate (Takara Bio, Otsu, Shiga, Japan), RNAiso Plus (Takara Bio), RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), and RNase-free DNase set (Qiagen) according to the manufacturer’s instructions. Frozen powdered tissue (50 mg) was suspended in 1 ml of Fruit-mate and centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was mixed with an equal volume of RNAiso Plus, incubated at room temperature for 5 min, mixed with 0.2 volumes of chloroform, and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous top layer was mixed with 0.5 volumes of ethanol, transferred into an RNeasy spin column, and centrifuged at 8000 × g for 15 s at room temperature; the flow-through was discarded. The subsequent steps for total RNA extraction were conducted using an RNeasy Plant Mini Kit and RNase-free DNase set. First-strand cDNA was
synthesized from 1.5 μg of total RNA using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with diluted cDNA (1:50) using a StepOne real-time PCR system and Fast SYBR Green system (both from Applied Biosystems) with the primer sets listed in Supplementary Table S1 and 18S rRNA as an internal control. Absolute mRNA copy numbers per pg of total RNA were calculated using standard plasmids carrying each aquaporin cDNA as reported by Sakurai-Ishikawa et al.16) Immunoblotting. Developing grains collected with the palea and lemma were immediately frozen in liquid nitrogen and ground with a mortar and pestle. Frozen powder was mixed with 1% (w/v) SDS, 2 mM EDTA, 20 mM Tris–HCl (pH 7.6), centrifuged at 13,000 × g for 10 min, and the supernatants were used as total protein for immunoblotting. SDS-PAGE was performed in 12.5% polyacrylamide gels, and proteins were transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA) in a semi-dry blotting apparatus for 40 min at 15 V. The membrane was blocked with 4% (w/v) skim milk in Tris-buffered saline and incubated with aquaporin antibody (1:1000 dilution). Anti-OsPIP2;1 and anti-OsTIP3;1 antibodies were raised against peptides MGKDEVMESGGAAGEFAAKDY14) and MSTAAARPGRRFTVGRS, respectively. Signals were visualized using horseradish peroxidase-linked Protein-A (1:2000 dilution) (GE Healthcare BioSciences, Piscataway, NJ, USA) and ECL detection reagents (GE Healthcare BioSciences).
High-temperature treatment. Temperature treatment was conducted during 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH in a growth chamber at 25/ 20 °C (control temperature treatment [CT]) or 32/27 °C (High-temperature treatment [HT]) day/night cycles. During grain filling, developing grains were collected and removed from the palea and lemma, and the fresh weight (FW) of each grain was measured immediately. Dry weight (DW) of developing grains was measured after oven-drying for more than 72 h at 80 °C. The relative moisture content in grains was evaluated from the ratio between fresh and DWs as (FW − DW)/ FW × 100. The moisture content of developing grains was evaluated from the difference between fresh and DWs (FW − DW). Immunohistochemical fluorescence staining. The 12-DAH grains were fixed in a solution containing 4% (w/v) paraformaldehyde, 0.08% glutaraldehyde, 5.0% acetic acid, and 50% ethanol. The fixed grains were first dehydrated through a graded ethanol series, then cleared with xylene, embedded in paraffin blocks, and sectioned vertically into 30-μm slices on a Leica RM2125RT microtome (Leica Microsystems GmbH, Nussloch, Germany). The 22-DAH or fully mature grains were cut vertically into halves with a razor blade, fixed in 4% (w/v) paraformaldehyde and 0.08%
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glutaraldehyde in 100 mM sodium phosphate buffer (pH 7.2), and dehydrated through a graded ethanol series. Grain sections and halves were blocked with 1% (w/v) bovine serum albumin in phosphate-buffered saline and 0.2% (w/v) Tween-20 and incubated with anti-aquaporin antibodies or pre-immune serum (1:100 dilution). After washing, Alexa Fluor 555 goat anti-rabbit IgG (1:200 dilution) (Invitrogen, Carlsbad, CA, USA) was used for visualizing aquaporins. Fluorescence was observed with an IX81 microscope equipped with a U-LH75XEAPO xenon burner and U-MWIG3 mirror unit (Olympus, Tokyo, Japan). Expression of rice aquaporins in yeast and determination of membrane osmotic water permeability. Rice aquaporin cDNA fragments were inserted into a yeast expression vector37,38) and expressed in Saccharomyces cerevisiae strain BJ5458 as described previously.12) Crude yeast membrane fractions were prepared according to the method reported previously.14) Membrane osmotic water permeability was measured by the stopped-flow light scattering method as described previously (stopped-flow spectrophotometer model SX18MV; Applied Photo Physics, Surrey, UK).14) Yeast membrane vesicles in 200 mM mannitol solution were quickly mixed with an equal volume of 400 mM mannitol solution. The mannitol solution contained 200 mM or 400 mM mannitol, 90 mM KCl, 1 mM EDTA, 1 mM EGTA, and 20 mM Tris–HCl, pH 7.2. Vesicle shrinking was monitored at 10 °C as an increase in scattered light intensity. Water transport activity was evaluated as membrane osmotic water permeability. Osmotic water permeability coefficient (Pf) was calculated from the initial rate constants (k), which were calculated from exponential fitting: Pf = (V0 × k)/(S × VW × Cout). The initial vesicle volume (V0) and the initial vesicle surface area (S) in 200 mM mannitol were calculated to be 7.63 × 10−3 μm3 and 1.87 × 10−1 μm2, respectively, according to the yeast membrane vesicle diameter in 450 mM mannitol.38) The partial molar volume of water (VW) was set as 18 cm3 mol−1. The initial concentrations of total solute outside the vesicle (Cout) were calculated from the concentration of 400 mM mannitol solution. The relative level of each aquaporin protein was calculated from signal intensity upon blot staining with anti-myc antibody using CS Analyzer ver 3.0 (ATTO, Tokyo, Japan).
Fig. 1. Dynamic changes in aquaporin expression levels and grain morphology during the grain filling period. Notes: The areas between the lines show the expression levels of each gene. Values are means of three independent growth experiments. To collect the grains, 1 tiller per plant was taken from at least three plants every 5 days. The 3rd to the 5th grains from the top on the 1st to the 3rd primary branches were selected. The transcript levels were normalized to the amounts of 18S rRNA. Aquaporins with 0.25 mRNA copies per pg total RNA) at the flowering stage (0 DAH, whole pistil). In the mid-grain filling stage, only several aquaporin transcripts, including OsPIP2;1, OsTIP2;2, and OsTIP3;1 were abundant (Fig. 1). Among them, OsPIP2;1 and OsTIP3;1 transcripts were predominant and peaked at 15 and 20 DAH, respectively (Fig. 1, see also Fig. 4). These two aquaporin
Fig. 2. Dynamic changes in the protein levels of OsPIP2;1 (A) and OsTIP3;1 (B) in developing grains. Note: Total protein (5 µg per lane) was subjected to immunoblotting with anti-aquaporin antibodies.
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(Fig. 2). Thus, the changes in the abundance of these two aquaporins were similar at the protein level.
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Effect of high temperature on dry matter accumulation, moisture content, and aquaporin expression in developing grains To evaluate the effect of high air temperature on dry matter accumulation and relative moisture content during grain filling, we measured fresh and DWs of the grains at different time points. Under CT, both parameters increased during the period from 5 to 20 DAH:
FW from 3.3 to 27.6 mg, and DW from 0.6 to 19.6 mg (Fig. 3(A)). A similar increase was observed under HT: FW increased from 3.3 to 23.3 mg and DW increased from 0.6 to 15.1 mg (Fig. 3(A)). During the same period, the relative moisture content decreased rapidly in both treatments and reached 28.9% in CT, whereas in HT, it remained 6.4 percentage points higher at 20 DAH (Fig. 3(B)). From 20 DAH, the relative moisture content stabilized in both treatments. At 45 DAH, the relative moisture content was 9.7 percentage points higher in HT than in CT (Fig. 3(B)). From 5 to 10 DAH, a rapid increase was observed in the moisture content per grain (Fig. 3(C)), indicating high water influx in this stage. In the mid-grain filling stage, the moisture content per grain decreased, indicating water efflux from the developing grain in this stage (Fig. 3(C)). The moisture content per grain was higher in HT than in CT from 30 DAH (Fig. 3(C)). We examined the effect of high air temperature on the gene expression levels of the two major aquaporins of developing grains. HT inhibited the increase in OsPIP2;1 expression during the period from 10 to 20 DAH (Fig. 4(A)) but did not affect that of OsTIP3;1 (Fig. 4(B)). Immunohistochemistry of aquaporins in rice grains In the 12-DAH grain sections, OsPIP2;1 was detected in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer (Fig. 5(A) and (D)). OsTIP3;1
Fig. 3. Effect of high temperature on FW and DW (A), the relative moisture content (B), and the moisture content per grain (C) in developing grains. Notes: Temperature treatment was conducted for 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH (time between the two broken lines) in a growth chamber at 25/20 °C (CT) or 32/27 °C (HT) day/ night cycles. Values are means ± SE of three independent growth experiments. Statistically significant differences between CT and HT at each DAH (Student’s t-test) are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4. Effect of high temperature on the expression of OsPIP2;1 (A) and OsTIP3;1 (B) in developing grains. Notes: Treatment was conducted for 2 weeks from 13:00 on 7 DAH to 13:00 on 21 DAH (time between the two broken lines) in a growth chamber at 25/20 °C (CT) or 32/27 °C (HT) day/night cycles. Values are means ± SE of three independent growth experiments. The transcript levels were normalized to the amounts of 18S rRNA. **p < 0.01, statistically significant differences between CT and HT at each DAH (Student’s t-test).
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Fig. 5. Localization of OsPIP2;1 and OsTIP3;1 in rice grains. Notes: The 12-DAH grain sections, and halves of 22-DAH and fully mature grains were labeled with respective anti-aquaporin antibodies and visualized with secondary antibody conjugated with Alexa Fluor 555 (red). AL, aleurone layer; DV, dorsal vascular bundles; NE, nucellar epidermis; NP, nucellar projection; PH, phloem; SE, starchy endosperm; XV, xylem vessels. Scale bars, 100 μm.
was detected in the aleurone layer and starchy endosperm, but not in the nucellar projection, nucellar epidermis, and dorsal vascular bundles (Fig. 5(B) and (E)). In the 22-DAH grains, OsPIP2;1 was detected mainly in the starchy endosperm (Fig. 5(F)), whereas OsTIP3;1 was found mainly in the aleurone layer (Fig. 5(G)). In the fully mature grains (from 45 DAH), OsTIP3;1 localization was similar to that in the 22DAH grains (Fig. 5(J)), whereas OsPIP2;1 staining became weak and difficult to detect (Fig. 5(I)).
Water transport activity of OsTIP3;1 We compared water transport activity of OsTIP3;1 and used OsPIP2;5 which has high water transport activity12,14) as a positive control and the vector as a negative control. The membrane fractions prepared from the yeast cells expressing rice aquaporins showed immunostained bands close to the expected molecular sizes of aquaporin monomers and dimers (Fig. 6(A)). The water transport activity of OsPIP2;5 was approx. 2.5 times that of the vector control. The water transport activity of OsTIP3;1 was similar to that of the vector control (Fig. 6(B)).
Discussion In this study, we investigated the expression of all 33 rice aquaporin genes in developing grains and found that only OsPIP2;1 and OsTIP3;1 transcripts were highly abundant in the mid-grain filling stage. Our data suggest that these two aquaporins might play different roles. OsPIP2;1 in grain filling Our study is the first to address the importance of OsPIP2;1 in grain filling. OsPIP2;1 was expressed
Fig. 6. The osmotic water permeability of yeast membrane vesicles. Notes: Crude membrane fractions prepared from yeasts expressing aquaporin constructs were subjected to immunoblotting with anti-myc antibody (A); 1 µg total protein was applied to each lane. Osmotic water permeability coefficient (Pf) of crude membrane fractions from cells producing recombinant OsPIP2;5 or OsTIP3;1, or cells carrying myc-pKT10 as vector control (B). Pf was corrected for the expression level of each aquaporin protein. The values are means ± SE of three individual replicates A–C (A). *p < 0.05, statistically significant difference from myc-pKT10 (Student’s t-test).
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Fig. 7. The proximal promoter region and 5′-UTR of OsTIP3;1. Note: ABRE-like and AW-box motifs are underlined. TSS, transcription start site. ATG, translation initiation codon.
abundantly in many organs, including developing grains (Fig. 1), leaves, and roots.12,18) From 5 to 10 DAH, OsPIP2;1 was expressed abundantly in developing grains together with several other aquaporins (Fig. 1). The water efflux rate was the highest during the period from 10 to 20 DAH (Fig. 3(C)), and at that time, OsPIP2;1 and OsTIP3;1 were most abundant (Figs. 1 and 4). Thus, we hypothesized that OsPIP2;1, OsTIP3;1, or both are important for net water efflux, dry matter accumulation, or both in developing grains after the mid-grain filling stage. Using the stopped-flow light scattering method, we have previously found that water transport activity of OsPIP2;1 was high.14) OsPIP2;1 was detected in many grain tissues (Fig. 5(A), (D), (F) and (I)), indicating its involvement in the cell-to-cell water transport pathway. We found that HT abolished the dynamic up-regulation of OsPIP2;1 during the mid-grain filling stage (Fig. 4(A)). Simultaneously, HT suppressed dry matter accumulation (Fig. 3(A)). OsPIP2;1 was located in the dorsal vascular bundles where the phloem and xylem vessels are located (Fig. 5(A) and (D)). OsPIP2;1 may enhance the phloem flow by discharging water from the phloem to the xylem. We propose that abolishing the dynamic up-regulation of OsPIP2;1 during the mid-grain filling stage may suppress dry matter accumulation under HT. High temperature during grain filling decreases rice grain weight and quality.39) Various mechanisms, such as changes in gene expression40,41) and nitrogen availability to rice plants,42) have been suggested to explain these effects. A decrease in water permeability due to the decreased OsPIP2;1 expression may be one of these mechanisms. Ishimaru et al. found that loose packing of grain amyloplasts leads to a higher moisture content under high temperature.43) Their observation is supported by our data that the relative water content in grains was higher in HT than in CT (Fig. 3(B)). OsTIP3;1 in grain filling The OsTIP3;1 transcript level from 15 to 25 DAH was not affected by HT (Fig. 4(B)), although dry matter accumulation during this period was reduced by HT (Fig. 3(A)). OsTIP3;1 had low water transport activity (Fig. 6(B)). These results suggest that OsTIP3;1 does not contribute considerably to the water flux, and HT induced decrease in the dry matter accumulation in the grain. Using immunohistochemical analysis, we detected OsTIP3;1 mainly in the aleurone layer in the 12-DAH, 22-DAH, and fully mature grains (Fig. 5(B), (E), (G) and (J)), whereas OsPIP2;1 was not observed
in the aleurone layer of the 12-DAH grains (Fig. 5(A) and (D)). These results suggest that the aleurone layer has low water permeability. For efficient assimilate transport, prompt absorption of water from phloem sap to xylem vessels may be needed.2–4) We believe that, to control water flow direction, the 12-DAH grains maintain high water permeability in the dorsal vascular bundles and low water permeability in the aleurone layer. Among all rice aquaporin genes, only OsTIP3;1 has 2 ABRE44)-like sequences located 200 bp upstream of the transcription start site in the proximal promoter region (Fig. 7); this indicates that a temporal increase in the OsTIP3;1 transcript level during grain filling might be mediated by ABA signaling. The aleurone layer accumulates triacylglycerols in oil bodies and phytin in the aleurone grains45) during the same stage of grain filling as when OsTIP3;1 is expressed. OsTIP3;1 has AW-boxes46) in the proximal promoter region (Fig. 7). The transcription factor WRINKLED1, required for triacylglycerol accumulation, binds to AWboxes. Thus, OsTIP3;1 may be involved in some of these metabolic pathways. In the present study, we did not examine the role of TIP3 phosphorylation in the regulation of water transport activity.34) We cannot rule out the possibility that OsTIP3;1 has water transport activity on another experimental condition or in planta. Further studies are needed to reveal OsTIP3;1 functions.
Supplementary material The supplementary material for this paper is available at http://dx.doi.org/10.1080/09168451.2015. 1032882.
Acknowledgments We are grateful to Prof. Masayoshi Maeshima for providing aquaporin antibodies and for valuable discussions throughout this work. We are grateful to Prof. Maki Katsuhara for valuable suggestions throughout this work.
Disclosure statement No potential conflict of interest was reported by the authors.
Aquaporins in developing rice grains
Funding This work was partially supported by the Bio-oriented Technology Research Advancement Institute (Brain) of Japan [Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) to M.M.]; the NARO Gender Equality Program.
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