Aquaporins in developing rice grains.

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.

Influence of low air humidity and low root temperature on water uptake, growth and aquaporin expression in rice plants.

The effects of low air humidity and low root temperature (LRT) on water uptake, growth and aquaporin gene expression were investigated in rice plants. The daily transpiration of the plants grown at low humidity was 1.5- to 2-fold higher than that at high humidity. LRT at 13°C reduced transpiration, and the extent was larger at lower humidity. LRT also reduced total dry matter production and leaf area expansion, and the extent was again larger at lower humidity. These observations suggest that the suppression of plant growth by LRT is associated with water stress due to decreased water uptake ability of the root. On the other hand, the net assimilation rate was not affected by low humidity and LRT, and water use efficiency was larger for LRT. We found that low humidity induced coordinated up-regulation of many PIP and TIP aquaporin genes in both the leaves and the roots. Expression levels of two root-specific aquaporin genes, OsPIP2;4 and OsPIP2;5, were increased significantly after 6 and 13 d of LRT exposure. Taken together, we discuss the possibility that aquaporins are part of an integrated response of this crop to low air humidity and LRT.

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.

Transpiration from shoots triggers diurnal changes in root aquaporin expression.

Root hydraulic conductivity (Lp(r)) and aquaporin amounts change diurnally. Previously, these changes were considered to be spontaneously driven by a circadian rhythm. Here, we evaluated the new hypothesis that diurnal changes could be triggered and enhanced by transpirational demand from shoots. When rice plants were grown under a 12h light/12h dark regime, Lp(r) was low in the dark and high in the light period. Root aquaporin mRNA levels also changed diurnally, but the amplitudes differed among aquaporin isoforms. Aquaporins, such as OsPIP2;1, showed moderate changes, whereas root-specific aquaporins, such as OsPIP2;5, showed temporal and dramatic induction around 2h after light initiation. When darkness was extended for 12h after the usual dark period, no such induction was observed. Furthermore, plants under 100% relative humidity (RH) showed no induction even in the presence of light. These results suggest that transpirational demand triggers a dramatic increase in gene expressions such as OsPIP2;5. Immunocytochemistry showed that OsPIP2;5 accumulated on the proximal end of the endodermis and of the cell surface around xylem. The strong induction by transpirational demand and the polar localization suggest that OsPIP2;5 contributes to fine adjustment of radial water transport in roots to sustain high Lp(r) during the day.

Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene.

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [(2)H(5)] and [(13)C(10)(15)N(2)]IAOx and [(13)C(6)], [(2)H(5)] and [2',2'-(2)H(2)]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2',2'-(2)H(2)]IAN overestimated the amount of IAN by 2-fold compared to when [(2)H(5)]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [(13)C(1)]IAN underestimated the amount of IAN when the ratio of [(13)C(1)]IAN standard to endogenous IAN was greater than five to one, whereas either [(2)H(5)]IAN or [(13)C(6)]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection∼100 pg/gfr.wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 µg/gfr.wt. IAN levels in these lines ranged from 0.6 to 6.7 µg/gfr.wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/gfr.wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.

Effect of low root temperature on hydraulic conductivity of rice plants and the possible role of aquaporins.

The role of root temperature T(R) in regulating the water-uptake capability of rice roots and the possible relationship with aquaporins were investigated. The root hydraulic conductivity Lp(r) decreased with decreasing T(R) in a measured temperature range between 10 degrees C and 35 degrees C. A single break point (T(RC) = 15 degrees C) was detected in the Arrhenius plot for steady-state Lp(r). The temperature dependency of Lp(r) represented by activation energy was low (28 kJ mol(-1)) above T(RC), but the value is slightly higher than that for the water viscosity. Addition of an aquaporin inhibitor, HgCl(2), into root medium reduced osmotic exudation by 97% at 25 degrees C, signifying that aquaporins play a major role in regulating water uptake. Below T(RC), Lp(r) declined precipitously with decreasing T(R) (E(a) = 204 kJ mol(-1)). When T(R) is higher than T(RC), the transient time for reaching the steady-state of Lp(r) after the immediate change in T(R) (from 25 degrees C) was estimated as 10 min, while it was prolonged up to 2-3 h when T(R) < T(RC). The Lp(r) was completely recovered to the initial levels when T(R) was returned back to 25 degrees C. Immunoblot analysis using specific antibodies for the major aquaporin members of PIPs and TIPs in rice roots revealed that there were no significant changes in the abundance of aquaporins during 5 h of low temperature treatment. Considering this result and the significant inhibition of water-uptake by the aquaporin inhibitor, we hypothesize that the decrease in Lp(r) when T(R) < T(RC) was regulated by the activity of aquaporins rather than their abundance.

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.

Isolation of chromosaponin I-specific antibody by affinity chromatography.

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.

Triterpenoid saponins stimulate the sugar taste receptor cell through a G protein-mediated mechanism in the blowfly, Phormia regina.

The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Previously we introduced chromosaponin I (CSI) and glycyrrhizin (GL), as sweet substances for the blowfly, Phormia regina. Application of these triterpenoid saponins induced feeding responses as well as impulses of the sugar taste receptor cell in the LL-type sensillum at a much lower concentration than that of sucrose. In the present paper, we show the involvement of G protein-mediated cascade in the CSI- and GL-responses as well as in sugar responses. CSI activates the sugar signal transduction cascade after penetrating through the membrane. On the other hand, GL exerts dual effects to stimulate the sugar signal transduction possibly by activating it inside the cell and also by interacting with the pyranose sugar receptor site. A non hydrolyzable G protein inhibitor guanosine 5'-O-(2-thiodiphosphate), GDPbetaS, markedly decreased the responses of the sugar receptor cell to the two triterpenoid saponins as well as the response to sucrose and fructose. These results suggest that CSI and GL are direct activators of G protein.