Transcriptome dynamics of rice in natura: Response of above and below ground organs to microclimate.

The long-term dynamics of the transcriptome under natural field conditions remain unclear. We conducted comprehensive gene expression analyses of rice leaves and roots grown under natural field conditions for a long period, from the tillering stage to the ripening stage. In this experiment, changes in the transcriptome were captured in relation to microclimatic parameters, particularly potential evaporation (Ep), which is a multiple meteorological factor and acts as an indicator of transpirational demand. The results indicated  that many genes were regulated by changes in temperature and Ep in both leaves and roots. Furthermore, the correlation between gene expression and meteorological factors differed significantly between the vegetative and reproductive stages. Since Ep triggers transpiration, we analyzed aquaporin gene expression, which is responsible for water transport, and found that many aquaporin genes in leaves were positively correlated with Ep throughout the growth period, whereas in roots, two plasma membrane intrinsic aquaporins, PIP2;4 and PIP2;5 were strongly correlated with Ep during reproductive growth. Other genes closely related to productivity, such as those involved in nutrient absorption and photosynthesis, exhibited different responses to meteorological factors at different growth stages. The stage-dependent shift in the microclimate response provides an important perspective on crop physiology in light of climate change.

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.

Vacuolar proton pumps and aquaporins involved in rapid internode elongation of deepwater rice.

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H(+)-ATPase (V-ATPase), H(+)-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.

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.

Osmotic water permeability of plasma and vacuolar membranes in protoplasts II: theoretical basis.

Water permeability of the plasma membrane (PM) and the vacuolar membrane (VM) is important for intracellular and transcellular water movement in plants, because mature plant cells have large central vacuoles. We have developed a new method for measuring the osmotic water permeability of the PM and VM (P ( f1) and P ( f2), respectively) in individual plant cells. Here, the theoretical basis and procedure of the method are discussed. Protoplasts isolated from higher plant tissues are used to measure P ( f1) and P ( f2). Because of the semi-permeability (selective permeability) of cellular membranes, protoplasts swell or shrink under hypotonic or hypertonic conditions. A theoretical three-compartment model is presented for simulating time-dependent volume changes in the vacuolar and cytoplasmic spaces in a protoplast during osmotic excursions. The model describes the theoretical relationships between P ( f1), P ( f2) and the bulk osmotic water permeability of protoplasts (P ( f(bulk))). The procedure for measuring the osmotic water permeability is: (1) P ( f(bulk)) is calculated from the time when half of the total change in protoplast volume is completed, by assuming that the protoplast has a single barrier to water movement across it (two-compartment model); (2) P ( f2) of vacuoles isolated from protoplasts is obtained in the same manner; and (3) P ( f1) is determined from P ( f(bulk)) and P ( f2) according to the three-compartment model. The theoretical relationship between P ( fl ) (m s(-1)) and L ( Pl ) (hydraulic conductivity, l=1, 2) (m s(-1) Pa(-1)) is also discussed.

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I: high osmotic water permeability in radish (Raphanus sativus) root cells as measured by a new method.

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.