High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.     

Differential responses of maize MIP genes to salt stress and ABA

Salt stress is known to reduce root hydraulic conductivity and growth. To examine a concomitant regulation of aquaporins, the expression of the maize MIP gene family in response to NaCl was analysed by DNA array hybridization. Plants responded differentially to 100 versus 200 mM NaCl treatments. Leaf water content was reduced rapidly and persistently after the application of 200 mM NaCl in contrast to 100 mM NaCl. Endogenous ABA strongly accumulated in roots after 2 h; it remained at a highly elevated level for 48 h after the addition of 200 mM NaCl, but rapidly declined in plants treated with 100 mM NaCl, indicating an early recovery from water deficit. Interestingly, 2 h after the addition of 100 mM NaCl, when maize regained the osmotic potential allowing water uptake, three highly expressed, specific isoforms ZmPIP1;1, ZmPIP1;5, and ZmPIP2;4 were transiently induced. They were preferentially transcribed in the outer root tissue suggesting a role in cellular water transport. None of the ZmTIP genes was altered. By contrast, after the addition of 200 mM NaCl these responses were missing. Instead, multiple ZmPIP and ZmTIP genes were repressed by 200 mM NaCl after 24 h. After 48 h, deregulations were overridden in both cases indicating homeostasis. ABA (1 muM) exogenously applied to the roots transiently induced ZmPIP2;4 similar to 100 mM NaCl as well as ZmPIP1;2. Thus, the early induction of ZmPIP2;4 by NaCl may be mediated by ABA. Previously, an increase in root hydraulic conductivity had been observed upon ABA application. By contrast, 100 muM ABA led to a complete, possibly non-specific repression of all detected ZmPIP and ZmTIP genes after 24 h.     

Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.     

Molecular mechanisms of urea transport in plants

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and “in-silico analysis”, two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.     

Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation

Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice ( Oryza sativa ) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 ( OsPT2 ) and OsPht1;6 ( OsPT6 ). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant.     

Selective Inhibition of Absorption and Long Distance Transport in Relation to the Dual Mechanisms of Ion Absorption in Maize Seedlings

The influence of several uncouplers of oxidative phosphorylation and inhibitors of terminal electron transport was studied on absorption and long distance transport of both K and C1 at concentrations within each range of the dual isotherm typical of ion uptake by maize roots. At low concentrations in the range of system 1, the system considered to implement ion movement through the plasma membrane, root absorption and long distance transport are equally inhibited by a given inhibitor. In the high range of system 2, the system considered to mediate ion passage through the tonoplast, long distance transport is markedly less sensitive to inhibitors than is absorption. The observations are in accord with the hypothesis that only system 1 is involved in the uptake of ions from the external solution into the symplast, and hence into the xylem. At high concentrations, entrance into the symplasm is deemed to be largely by diffusion and therefore less inhibitor sensitive.With respect to absorption by the roots, the plasma membrane system is more inhibitor sensitive than is the tonoplast system. It is suggested that the difference in sensitivity is real, and not the consequence of an inequality of inhibitor concentration in the vicinity of the plasma membrane and tonoplast respectively.     

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

 Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity. Received: 2 July 1998 / Accepted: 14 September 1998     

Phosphorus effect on the uptake,translocation and accumulation of the14C-urea in orchard grass (Dactylis glomerata L.)

Summary The effect of different phosphorus supplies on the uptake, translocation and accumulation of¹⁴C-urea by orchard grass was investigated. Phosphorus starvation inhibits the uptake, translocation and accumulation of the carbon of urea similarly to the nitrogen of urea. As compared with the uptake process the reduction of the accumulation is much more effected by the inhibition of the carbon translocation from roots to the aboveground parts. Lack of phosphorus also decreases the incorporation of the¹⁴C of urea into high-molecular compounds. The effect of phosphorus deficit on the accumulation of¹⁴C-urea increases with time of starvation.     

AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots

Despite the fact that urea is a ubiquitous nitrogen source in soils and the most widespread form of nitrogen fertilizer used in agricultural plant production, membrane transporters that might contribute to the uptake of urea in plant roots have so far been characterized only in heterologous systems. Two T-DNA insertion lines, atdur3-1 and atdur3-3, that showed impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transporter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing AtDUR3-promoter:GFP constructs, promoter activity was upregulated under nitrogen deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. Protein gel blot analysis of two-phase partitioned root membrane fractions and whole-mount immunolocalization in root hairs revealed the plasma membrane to be enriched in AtDUR3 protein. Expression of the AtDUR3 gene in nitrogen-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher accumulation of urea in roots of wild-type plants relative to atdur3-1 and atdur3-3 confirmed that urea was the substrate transported by AtDUR3. Influx of 15N-labeled urea in atdur3-1 and atdur3-3 showed a linear concentration dependency up to 200 microM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 microM. The results indicate that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggest that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils.     

全膜双垄沟覆盖条件下不同施氮量对春玉米氮素吸收利用及分配的影响

通过田间试验研究了黄土旱塬旱作全膜双垄沟覆盖栽培条件下,不同施氮量（0、100、200、250、300及400 kg/hm²）对春玉米氮素吸收、利用及分配的影响,为提高春玉米氮素利用效率及合理施氮提供理论依据。结果表明:（1）春玉米植株及籽粒含氮量、氮素累积量随施氮量增加而提高,但当施氮量超过250 kg/hm²后增加效果不显著。（2）春玉米植株含氮量随生育期推进而降低,但氮素累积量则随生育期推进而增加。（3）玉米叶片及茎+叶鞘氮素转移量及对籽粒氮素贡献量高于其他器官。（4）春玉米籽粒产量随施氮量增加先增加后降低,并在施氮量为250 kg/hm²时产量最大（11 932 kg/hm²）,此时氮素收获指数最大（69.12%）并显著高于其余处理,氮肥农学效率也显著高于施氮量为300、400 kg/hm²的处理。因此,从春玉米产量、氮素利用角度考虑,该试验条件下的合理施氮量为250 kg/hm²。     

Cadmium uptake,localization and detoxification in <Emphasis Type="Italic">Zea mays</Emphasis>

Cadmium uptake, translocation and localization in maize roots and shoots at the tissue and cellular level were investigated. Metal accumulation in plant organs as well as symptoms of Cd toxicity were closely correlated with an increase in Cd concentration applied (5 – 300 M). Most of the metal taken up was retained in roots, mainly inside the cells of endodermis, pericycle and central cylinder parenchyma. Accumulation of phytochelatins and related peptides also depended on Cd concentration in the nutrient solution.     

Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis

Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP), is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.