hydroponic resources - plant grow & hydroponic nutrients solutions

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Australia's online hydroponics supplies & indoor grow shop

Hydroponic plants do not grow in soil. The roots find their home in water or an inert, solid medium such as vermiculite or sand, which serve merely to support the plant.

A hydroponic setup can be put together at home with a wide-mouth jar filled with a nutrient solution, a stopper and an aquarium aerator. The stopper needs two holes, one for a tube from the aerator and a larger hole for the plant's stem. Wedge some cotton in the larger hole to keep the plant from slipping down into the drink. Wrap the jar in aluminum foil to exclude light.

To grow plants in an inert, solid medium, you need two watertight containers with spouts at their bottoms joined by a flexible plastic hose. One container is for plants to grow in the medium. The other is for a nutrient solution. You must lift the container with the nutrient solution just high enough and just long enough to raise the nutrient solution around plant roots in the other container.

Raising and lowering a container of nutrient solution a half- dozen times per day (which might be needed for a rapidly growing plant) can get tedious. The process can be automated by having the nutrient reservoir set permanently below the plants, with a time- activated pump to feed the plants. Another way to get nutrient solution to the plant would be with a thick polyester rope wick. Put one end in a nutrient reservoir just below the plants and the other end up through the bottom of the plant container, with the ropes end unraveled and fanned out in the growing medium.

Make up a simple nutrient solution by dissolving a soluble fertilizer (look for a complete fertilizer with micronutrients) in water. Organic fish emulsion fertilizer is another option.

Some experimentation is needed to get the right nutrient balance to adequately nourish plants. Off-color leaves are signs that a plant needs more fertilizer. Dry leaf margins indicate too much fertilizer.

Needs will vary with the kind of plant being grown as well as growing conditions.

A gardener might want to try hydroponics because it's yet another fun way to fiddle around with plants. Those gardeners who are mechanically inclined might also like applying those skills to automating their hydroponic setups.

Commercially, the advantages of hydroponics are potential water savings and the ability to more completely automate plant care.

The Need for Ph

Agriculture: Proper soil pH ensures maximum crop yield.
Drinking Water: Rigid purification standards depend upon pH measurement and control.
Sewage Treatment: Successful treatment depends on pH control throughout the process.
Industrial Process: Petrochemical, pulp and paper, food and beverage, refining, power generation, pharmaceutical, semiconductor, automotive, steel and metal processing; all require pH measurement and control for product quality and efficiency.
Environmental: pH monitoring and control are critical to the continuation and quality of all plant and animal life.

What are the mineral elements ?

There are twenty mineral elements considered necessary or beneficial for plant growth. Carbon (C), hydrogen (H), and oxygen (O) are supplied by air and water.

The six macronutrients, nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulphur (S) are required by plants in large amounts.

The rest are required in trace amounts (micronutrients). Essential trace elements include boron (B), chloride (Cl), copper (Cu), iron (Fe), manganese (Mn), sodium (Na), zinc (Zn), molybdenum (Mo), and nickel (Ni).

Beneficial mineral elements include silicon (Si) and cobalt (Co). The beneficial elements may only be essential for some plants. Cobalt for instance is essential for nitrogen fixation in legumes. It may also inhibit ethylene formation and extend the life of cut roses. Silicon, deposited in cell walls, has been found to improve heat and drought tolerance and increase resistance to insects and fungal infections. Excess silicon can cause strawberries to remain white when ripe. Silicon can help plants deal with toxic levels of manganese, iron, phosphorus and aluminium as well as zinc deficiency.

The most productive approach includes mineral elements at levels beneficial for optimum growth.

An typical nutrient formulation is listed below. To the right is the general range of nutrient elements used by plants. Part A and Part B refer to the requirement to keep particular nutrients separated in the concentrated form because of the fact that many nutrient combinations will precipitate. For instance Calcium Soleplate is plaster of Paris and will precipitate when calcium salts are mixed with soleplate salts in concentrated form.
Note: (1 mg/litre = 1 ppM)

As plants grow their requirements change and so the nutrient solution must also be changed to keep the plants growing at their optimum. Ammonia has an adverse effect and needs to be kept to a minimum.

Iron and Manganese ions oxidize and precipitate out of solution causing problems of staining and nutrient deficiency. This process is exacerbated when the water is sanitized with UV light or chemicals. Magnets in the nutrient flow will attract and remove Iron oxide (it is magnetic) keeping the plant root systems and sterilisers cleaner.

Plants use some nutrients like nitrates at a faster rate than other nutrients. This causes the solution pH to increase requiring the addition of acids like Phosphoric Acid or Nitric Acid.

Nutrient Management ideals

A major difficulty in using recirculating solutions is management of the nutrient balance. This is much more difficult than with non-recirculating systems.

Open systems do a run to waste with the nutrient solution. Closed systems collect the nutrient solution and reuse it. For closed systems a wide range of collection systems are in use, many developed and sold by supply companies. They can vary greatly in initial cost and no type yet dominates the market. The basis of the operation of a typical closed substrate system is as follows: about 30% excess is run off from the medium, collected in channels and stored in a tank. This is then pumped through a steriliser. The sterilised solution is mixed with fresh nutrient solution to make the feed to the drippers.

Once a week, the run-off solution is sampled for complete analysis. The analysis service includes advice on what fertiliser balance should be used for the next week. The fresh nutrient solution composition is changed to follow these recommendations. Fertilisers can be purchased in concentrated liquid form. Commercial growers like those in Holland have an eight tank system, into which the concentrated fertilisers are pumped. Supplying fertiliser in this form is possible only in Holland because of the compact nature of the industry. In other countries, the much smaller size of the industry and the distances involved in cartage, would make the cost of this method prohibitive.

Most growers still use A and B concentrated nutrient tanks, but using the individual liquid fertilisers makes changing formulations much easier. Using separate nutrient tanks makes it possible for nutrient formulation to be remotely controlled by computers.

Please notice - different crops, growing environments and growth stages will require different nutrient formulations.

It has been recognized for centuries that soils containing ample organic matter are typically more productive than those of a sandy nature. Organic matter can improve soil water holding capacity, cation exchange capacity, nutrient retention, soil microbial activity, and other soil properties (Stevenson, 1994). In recent years, plant scientists have begun to study specific components of soil organic matter to determine their influence on plant growth. In particular, much activity has focused on evaluating humic substances present in soil (MacCarthy et al., 1990).

Humic substances have been defined by Aiken et al. (1985) as "a category of naturally occurring, biogenic, heterogeneous organic substances that can generally be characterized as being yellow to black in color, of high molecular weight, and refractory". Humic substances can be divided into humic acids (HA), fulvic acids (FA), and humins based on their solubility in acid and alkali. Humic acid is produced by extracting an organic matter source with an acidic solution (pH = 2.0); any fraction which is insoluble below pH 2.0, but soluble above, is considered a humic acid.

The positive effect of humic substances on the growth of numerous plants in the Gramineae family has been well documented (Chen and Aviad, 1990). Dixit and Kishore (1967) reported enhanced germination in corn (Zea mays L.), barley (Hordeum vulgare L.), and wheat (Triticum aesitvum L.) treated with humic or fulvic acids. Kononova and Pankova (1950) compared the root development of corn growing in solution culture with or without added humic acid and found that root length and number doubled in response to humate added at 4 to 5 mg [L.sup.-1]. Lee and Bartlett (1976), also working with corn grown in solution culture, found that rooting was enhanced significantly at a humate concentration of 8 mg [L.sup.-1]. Vaughan and Malcolm (1985) compared root and shoot growth of wheat grown in water alone, a complete (Hoagland's)nutrient solution, and each solution supplemented with 50 mg [L.sup.-1] humic acid. Their experiments showed a 58% increase in root growth when humic acid was added to water alone. Addition of humic acid to plants growing in nutrient solution increased root growth approximately 25% compared with plants growing in nutrient solution alone.

In a review of research evaluating humic substances, Chen and Aviad (1990) cite many studies demonstrating the influence of humic materials on nutrient uptake by plants. Dormaar (1975) reported an increase in nitrogen (N) uptake by rough fescue (Festuca scabrella Torr.) in response to application of humic substances extracted from three soils while phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na) uptake were unaffected. Guar (1964) found increased N, P, and K uptake in perennial ryegrass (Lolium perenne L.) grown in sand amended with humic acid extracted from compost. Varshovi (1991) found no increase in N uptake of bermudagrass (Cynodon dactylon L.) following application of a commercial humate material at 0, 268, and 803 kg [ha.sup.-1]. Whether nutrient uptake increases, decreases, or remains constant in response to humic substances appears to depend in large part on the plant species and humic materials evaluated.

During recent years many commercial products containing HA have been promoted for use on turfgrasses. While the effects of humic substances on cereal grasses and numerous other plants have been well documented, the growth response of turfgrasses has not been studied extensively. The purpose of this work was to determine the potential of humic substances (including both humate and humic acids) to effect root growth and nutrient uptake of creeping bentgrass turf growing in sand or solution culture.

`Crenshaw' creeping bentgrass sod was harvested and trimmed of all roots before being transplanted into 15.2-cm diam. by 30.5-cm-deep pots in the greenhouse on 17 May 1996. The rooting media was a washed quartz sand containing 650 g [kg.sup.-1] medium, 210 g [kg.sup.-1] fine, and 120 g [k.sup.-1] coarse sand with a CEC of [is less than] 1.0 cmol [kg.sup.-1]. Treatments (Table 1) were initially applied on 24 May 1996 and included a commercial granular humate (715 g [kg.sup.-1] organic matter), a commercial soluble humic acid, and three standard humic acid materials extracted from soil, peat, or Leonardite (International Humic Substances Society, St. Paul, MN) and applied as solutions. Liquid treatments were applied with a [CO.sub.2]-powered sprayer at 207 kPa pressure delivering 2000 L [ha.sup.-1]. Granular humate was uniformly mixed into the upper 10 cm of granular-treated pots prior to sod placement. Liquid treatments were reapplied on 21 June and 16 August. The turf was mowed to 6 mm three times per week throughout the study with irrigation applied as needed to prevent drought stress. The experimental design was a randomized complete block with four replications.

Table 1. Source, formulation, application rate, and nutrient content of humic substances used in sand and solution culture.

([dagger]) Mention of a brand or trade name does not imply endorsement by North Carolina State University. Menefee humate (Earthgro Products Inc., Dallas, TX); Sustane HA (Sustane Corp., Cannon Falls, MN).

([double dagger]) (G) granular formulation incorporated into the top 10 cm of sand in the sand culture experiment only. (S) soluble formulation applied as a foliar spray in sand culture and solution-culture studies.

([sections]) Treatments in the sand-culture experiment were applied initially on 24 May 1996. Soluble materials were reapplied on 21 June and 16 Aug. 1996. In the solution-culture experiment, foliar application was carried out on 24 Jan., 7 Feb., 21 Feb., and 7 Mar, 1997.

Average maximum and minimum greenhouse temperatures during the study were 34 and 22 [degrees] C, respectively. One-quarter strength Hoagland's solution (Hoagland and Arnon, 1950) was applied every 3 d at 180 mL [pot.sup.-1], providing 50 kg N [ha.sup.-1] [month.sup.-1]. No pesticides were used during the course of the study.

Crenshaw creeping bentgrass sod was trimmed of all roots, cut into 10.8-cm-diam. plugs and transferred into two 126-[dm.sup.3] solution-culture tanks on 20 Nov. 1996. The tanks were located in a greenhouse with average maximum and minimum greenhouse temperatures of 31 and 21 [degrees] C, respectively, during the study. No supplemental lighting was necessary to maintain adequate growth. The pH of the culture solution was maintained at 6.0 [+ or -] 0.1 by automated additions of 100 mol [m.sup.-3] [H.sub.2] [SO.sub.4]. One-quarter strength Hoagland's solution was used to provide complete nutrition to the plants. The solution was continuously aerated and was completely replaced every 2 wk to assure an adequate nutrient supply.

Treatments (Table 1) were initially applied on 24 Jan. 1997 as described for the sand-culture experiment except that granular humate was not evaluated. Treatments were reapplied on 7 and 21 Feb. and 7 Mar. 1997. The turf was mowed to 6 mm three times per week throughout the study. The experimental design was a randomized complete block with five replications.

In order to assess the effect of humic substances on rooting, both root mass and root length were determined for sandculture and solution-culture experiments. Each plug and its associated root system were completely removed from its pot in the sand-culture experiment on 2 Oct. 1996. The sand was gently shaken from each root system before sectioning into 0- to 10-, 10- to 20, and [is greater than] 20-cm depths for root mass determination. Roots from each depth were thoroughly washed over a fine mesh screen to remove all sand and then were ovendried at 75 [degrees] C for 48 h before weighing to determine root mass. Preliminary research confirmed no significant difference between this method and ashing of the root system at 600 [degrees] C for 72 h. In the solution-culture experiment, the turf and its associated root system were harvested from each cell in the system on 28 Mar. 1997. After measuring root length, the root system of each plug was harvested and oven-dried at 75 [degrees] C for 48 h before weighing to determine root mass.

To determine if application of humic substances resulted in enhanced nutrient uptake, leaf tissue was harvested every 2 wk from each treatment. In both sand- and solution-culture experiments, because of the small area harvested, clippings from all harvest dates (2-wk intervals) were combined to produce a composite sample for analysis. Clippings were ovendried at 75 [degrees] C for 48 h and stored until analysis. Nitrogen was determined using a Perkin-Elmer PE2400 CHN Elemental Analyzer (Perkin-Elmer Corp., Norwalk, CT). A Perkin-Elmer Plasma 2000 (an inductively coupled plasma emission spectrometer) was used to determine P, K, Fe, Ca, and Mg following dry-ashing by a slight modification of the technique described by Grewling (1976). Sulfur was analyzed with a Perkin-Elmer Plasma 2000 inductively coupled plasma emission spectrometer according to the wet-ashing technique of Novozamsky et al. (1986).

All data were subjected to an analysis of variance (ANOVA) with the Statistical Analysis System (SAS Institute. Inc., 1985). Separation of significantly different treatment means was accomplished with pre-planned orthogonal contrasts (Steel and Torrie, 1980). The Waller-Duncan K ratio t-test (SAS Institute, Inc., 1985) was employed for mean separation if the ANOVA F-test indicated that source effects were significant.

Creeping bentgrass root mass and length in response to application of humic substances in sand culture is summarized in Table 2. Rooting responses did not vary with application rate for any humic substance source; thus, data are presented as the mean of all application rates for a given source. Planned contrasts revealed that, as a group, the humic substances (soil-incorporated granular and foliar-applied liquids) increased average root mass 26% in the upper 10 cm of rootzone compared with the control. This relationship also held true for rooting at a depth greater than 20 cm where root mass in response to humic substances averaged 0.13 g compared with 0.04 g for non-treated turf. Creeping bentgrass grown with soil-incorporated granular Menefee humate produced significantly greater root mass at the 0- to 10- cm depth than turf receiving any foliar HA application (Menefee humate vs. Foliar HA's contrast). At the 10- to 20-cm rooting depth, incorporated granular humate resulted in 29% greater root mass than turf receiving foliar peat HA or Sustane HA treatments. Incorporating granular humate provided root growth superior to the control at all rooting depths.

Table 2. Effect of humic substance application([dagger]) on the root mass and maximum root length of creeping bentgrass grown in sand-culture as determined on 20 Oct. 1996.

([dagger]) All treatments were applied initially on 24 May 1996. Soluble humic acids were reapplied on 21 June and 16 Aug. 1996.

([double dagger]) (G) granular formulation incorporated into the top 10 cm of sand. (S) soluble formulation applied as a foliar spray.

([sections]) Mean separation within columns by Waller-Duncan K ratio (K = 100) t-test. Means in the same column followed by the same letters are not statistically different at P = 0.05.

Foliar HA treatments had little effect on root mass with only foliar applied soil-extracted HA producing rooting superior to the control at the 0- to 10- and [is greater than] 20-cm depths. Leonardite HA increased root root mass 375% at the [is greater than] 20-cm depth compared with the control while peat HA and Sustane HA did not improve rooting at any depth in the sand-culture experiment.

Granular humate incorporation did not increase average maximum root length significantly compared with foliar applied HA sources (Table 2); however, granular application resulted in a maximum root length 15% greater than that of non-treated turf. Foliar HA application resulted in a mean maximum root length (34.3 cm) not significantly different from that of the control (32.2 cm).

In the solution-culture experiment (Table 3), foliar application of HAs did not result in improved root mass compared with untreated turf regardless of application rate. Planned contrasts revealed little difference in response among the various sources; however, the Leonardite-derived HA did result in a mean maximum root length 15% greater than that of the control and bentgrass treated with either soil or peat-derived HAs.

Table 3. Effect of humic acid application([dagger]) on root mass and maximum root length of creeping bentgrass grown in solution-culture as determined on 28 March 1997.

([dagger]) All treatments were applied as foliar sprays on 24 Jan., 7 Feb., 21 Feb., and 7 Mar. 1997.

([double dagger]) Meal separation within columns by Waller-Duncan K ratio (K = 100) t-test. Means in the same column followed by the same letters are not statistically different at P = 0.05. (*), NS, Significant at P = 0.05 and nonsignificant, respectively.

Nutrient concentration did not vary significantly with application rate for any of the humic substances; thus, data are presented as the mean of all application rates for a given source (Table 4). Nitrogen and calcium content of creeping bentgrass were unaffected by humic substance application and averaged 46.3 and 6.0 g [kg.sup.-1], respectively, across all treatments. Planned contrasts revealed that application of humic substances, averaged across all treatments, provided significantly greater tissue P concentration (6.43 g [kg.sup.-1]) than that of nontreated turf (6.20 g [kg.sup.-1]). Incorporated granular Menefee humate, as well as foliar applications of soil-derived HA, peat HA, and Leonardite HA resulted in an average leaf P content significantly greater than the control. Only Sustane HA failed to increase the P uptake of creeping bentgrass compared with the control.

Table 4. Effect of humic substance application[(dagger]) on the nutrient concentration of creeping bentgrass grown in sand-culture.

(*), (**), (***), NS, Significant at P = 0.05, 0.01, 0.001, and nonsignificant, respectively.

([dagger]) All treatments were applied initially on 24 May 1996 and humic acids were reapplied on June 21 and 16 Aug. 1996.

([double dagger]) Mean separation within columns by Waller-Duncan K ratio (k = 100) t-test. Means followed by the same letter are not statistically different at P = 0.05.

Potassium uptake was not greatly influenced by the humic materials with only Leonardite HA applications increasing tissue K concentration compared with untreated turf. Planned contrasts (Table 4) show that only incorporated Menefee humate resulted in an average Mg content greater than that of non-treated turf (3.15 g [kg.sup.-1] vs. 3.03 g [kg.sup.-1] for the control). The average tissue Mg concentration of plants treated with foliar HAs (3.10 g [kg.sup.-1]) was similar to that of plants receiving granular incorporated humate.

Of all elements evaluated, S was the only element whose uptake was diminished significantly by foliar application of humic substances. The S content of nontreated turf was approximately 7% greater than that of turf treated with foliar HAs, but was comparable to that of plants growing in incorporated granular humate, which had no effect on S uptake. As with tissue N and Ca content, Fe uptake was not significantly influenced by humic substance treatments.

As in the sand-culture experiment, humic acid application had no effect on creeping bentgrass N content with both treated and non-treated plants averaging 60.2 g N [kg.sup.-1] (Table 5). Contrary to the results noted in sand culture, in solution culture the average P content of treated turf did not vary significantly from that of the control.

Table 5. Effect of humic substance application([dagger]) on the nutrient concentration of creeping bentgrass grown in solution-culture.

(*), (***), NS, Significant at P = 0.05, 0.001, or nonsignificant, respectively.

([double dagger]) Mean separation within columns by Waller-Duncan K ratio (K = 100) t-test. Means followed by the same letter are not significantly different at P = 0.05.

The potassium concentration of plants treated with humic acids decreased approximately 7% compared with non-treated plants grown in solution culture (Table 5) in contrast with the sand-culture experiment where there was no meaningful effect on K uptake. The effect was very consistent and occurred with all treatments except soil HA at 100 mg [L.sup.-1] where no difference was noted. Calcium, Mg, S, and Fe content were not influenced appreciably by application of humic acids. While tissue nutrient content in response to a particular treatment occasionally varied significantly from non-treated turf, there were no notable trends in response to treatments.

The tendency for incorporated granular humate to induce significantly greater root mass than foliar HA applications in sand culture may have been a result of granular humate and its breakdown products being in more direct contact with roots compared with HA treatments which were applied to foliage. The granular humate used in this study had an organic matter content of 715 g [kg.sup.-1] and may have contributed to improved soil fertility in the rootzone via improved water retention in the rhizosphere as well as by improved nutrient retention. Considering the substantial cation exchange capacity of the incorporated Menefee humate product (61 cmol [kg.sup.-1]), the retention of nutrients from weekly Hoagland's solution applications may have been enhanced and facilitated improved root growth.

In addition to the indirect effects noted above, incorporating humate into the root zone may directly stimulate rooting via alteration of membrane characteristics or plant energy metabolism (Chen and Aviad, 1990). It has been suggested by Syltie (1985) that humic substances supply polyphenols that catalyze plant respiration. Increases in root growth might be attributed to enhancement of enzyme systems stimulated by increased respiration. Whatever the pathway for improved rooting, incorporation of the material was important in the process. Foliar applied materials did not improve rooting notably. Doter and Peacock (1997) also reported no improvement in rooting for a `Cato'/Crenshaw creeping bentgrass blend receiving foliar applications of humates.

Incorporated granular humate resulted in a maximum root length significantly greater than the control. As with root mass determinations, this may have been due to the proximity of the material to roots compared with foliarly applied HA treatments.

The lack of a notable rooting response for plants grown in solution culture compared with sand-cultured bentgrass may have been a result of greater nutrient availability in solution culture compared with the sand medium. Several researchers have attributed improved root growth associated with humic substances to increased P uptake (Chen and Aviad, 1990; Lee and Bartlett, 1976). In the sand-culture experiment, plants receiving an application of any of the humic substances averaged 4% greater P content than the control. Increased P uptake in response to humic substances is a fairly common response, having been previously reported in pot culture experiments for Italian ryegrass (Lolium multiflorum L.) (Brun et al., 1996) and perennial ryegrass (Guar, 1964). In solution culture, however, the average P content of treated turf (6.62 g [kg.sup.-1]) was no different than that of the control (6.68 g [kg.sup.-1]). Additionally, the P content of non-treated plants in solution culture was 8% greater than that of non-treated plants in sand culture. Thus, it is likely that in the solution-culture system, with optimum nutrient availability for all treatments including the control, any advantage potentially conferred by enhanced P uptake would be absent.

Potassium uptake was relatively unaffected by application of humic substances in sand culture while all humic acids reduced tissue K concentration compared with the control in solution culture. Dormaar (1975) reported no increase in K uptake of rough fescue grown in nutrient solution amended with humic acids extracted from three soils. Tan and Nopamornbodi (1979) likewise found no effect on the K content of corn treated with humic substances. Iron uptake in both sand and solution culture studies was also relatively unaffected by humic substance application. This was somewhat surprising since several researchers have reported increased shoot iron content in response to humic materials (Chen and Aviad, 1990; DeKock, 1955; Lee and Bartlett, 1976). Dorer and Peacock (1997) reported that applications of granular humate to creeping bentgrass resulted in higher tissue Fe levels than plots receiving liquid humate or the control. Perhaps providing Fe in these studies via foliar application of Hoagland's nutrient solution every 3 d (sand culture) or via Hoagland's solution (solution culture) obscured the development of potential tissue Fe differences. While most researchers evaluating plants other than turfgrass have noted increased N uptake with humic substance application (Chen and Aviad, 1990), the findings of no influence on N uptake in these studies corroborates work by Varshovi (1991) on bermudagrass and Dorer and Peacock (1997) working with creeping bentgrass who also reported no increase in N uptake by turf treated with humates.

The absence of significantly enhanced P uptake in solution-culture grown plants may have been due to a non-limiting pool of P available in the solution-culture system compared with the relatively infertile sand root-zone presented by sand culture. It has been suggested by Dormaar (1975) and Chen and Aviad (1990) that humic substances may have limited growth-promoting effect on plants adequately supplied with nutrients. Potassium levels in plants receiving HAs and growing in solution culture were significantly less than for control plants, whereas no difference in tissue K content was noted in sand-culture grown plants. It is difficult to understand why K uptake might decrease in response to humic acid application since the majority of research to date indicates either no effect or increased K uptake resulting from humic substance application.

While K uptake in response to HA treatment varied with growing medium (sand vs. solution culture) it is important to note that in both cases the level of tissue K was above the 22 to 26 g [kg.sup.-1] sufficiency level reported for creeping bentgrass (Mills and Jones, 1996). Indeed, accepted nutrient sufficiency levels were met or exceeded for all nutrients measured in both studies. When significant differences in nutrient concentration were observed among treatments, they were generally tess than 3 mg [kg.sup.-1] Dorer and Peacock (1997) also reported statistically significant but small differences in nutrient uptake due to humate application and questioned whether such differences were of biological significance.

In general, application of humic substances via foliar application had limited effect on the rooting or nutrient uptake of creeping bentgrass. Creeping bentgrass root mass at all depths of measurement was increased, however, by incorporation of granular humate into the top 10 cm of a sand rootzone prior to sod placement, perhaps due to more proximal contact with developing roots. No single foliar humic acid treatment consistently improved rooting compared with the control in either sand- or solution-culture experiments.

Nitrogen, Ca, Mg, and Fe concentration of leaf tissue were relatively unaffected by the application of humic substances, regardless of application rate. Phosphorous uptake in the sand-culture experiment was increased by incorporated granular humates as well as several foliar-applied humic substances. The influence of HA application on nutrition of solution-grown plants was minimal. The lack of improved rooting and P uptake in solution culture may support the hypothesis suggested by Dormaar (1975) and Chen and Aviad (1990) that humic substances have limited reduced growth-promoting effects on plants adequately supplied with nutrients.

Roses, shrubs and most other plants can suffer from a lack of certain nutrients and show symptoms similar to warm-blooded mammals like us. We call it anemia; in the plant world it's called chlorosis.

As a general rule, it appears in midsummer and it's on time again this year. As temperatures go up and heat and drought stress intensify, more will show up. Chlorosis is easy to identify. Leaves will turn pale green and, in severe cases, yellow to whitish. Veins in the leaves stay at full color, and as the leaf tissue becomes paler, they appear an even deeper green.

On roses, shrubs, trees and other leafy plants, the sickly color will usually be found on the youngest shoots. The same pale hue is evident on evergreen-branch tips, especially juniper. Growth on lawns slows, and the blades' normal green fades to an unhealthy cast.

The problem centres around the fact that while our soils have a high mineral content, including iron; many are in a form that plants can't use. The more alkaline a soil is (higher pH), the less iron is available. Copper, zinc and some other minor food elements react in the same way. Without these elements, even in small amounts, the food-making process (photosynthesis) slows or is put on hold. As the plant's defenses weaken, chances of disease and insect problems increase.

The obvious solution is to either lower the pH of the soil, introduce some usable form of needed trace elements, or both if possible. Iron is available on the retail market in two main forms. The first is iron soleplate as a whitish crystal that's water-soluble. It has a couple of drawbacks, however: It doesn't last long, and it can stain sidewalks and driveways. The second is a variety of iron forms that have been chelated, which is a fancy name for a treatment that allows iron to be available for a relatively long time.

Liquid irons are also on the market, and many are chelated to a degree. I prefer to use this form for spot treatments. It's easy to handle and dilutes in water, and results will often show up in just a few days. Small perennials, roses and shrubs will respond to some of the solution poured around their bases and then watered down. Large shrubs and trees do better if the iron solutions are put closer to their active root systems.

Make holes 6 to 18 inches deep around lilacs and up to 18 inches for mature trees. Active roots on trees start a couple of feet inside a line under the longest, lowest branches and go outward for many feet. Proper holes can be made with many tools, but hose-attached tools such as the Ross Root Feeder are the easy way. Ross also provides pre-measured tablets of iron that can be dissolved and injected into the soil through this tool.

Liquid iron mixed with water can also be poured into these holes, as can solutions of iron soleplate. If the plants haven't been fed over the past four to five weeks, place a balanced solution in every other hole, alternating with iron. The addition of sulphur will lower the alkalinity making iron, copper, zinc and manganese more available to the plant.

Annual applications of sulphur flakes or pellets, at five to eight pounds per 1,000 square feet of lawn, are a sounder approach than the ``one shot'' program. Either drop or rotary spreaders will do the job. A rotary is faster, and if some is thrown into vegetable or flower beds as you go by, so much the better. A couple of handfuls scattered around each rosebush once or twice a year also will help. Just scratch it in and water it down. Soil bacteria will eat and digest the sulphur particles, leaving it in a highly usable form.

In the fall, spread sulphur dust instead of pellets around the bases and out a foot or so from roses, perennial phlox and peonies. It suppresses many disease organisms and even knocks off some bug problems such as spider mites. As it filters down into the soil, it also will be broken down.

When selecting fertilizers, always go for those with sulphur and iron added. There's usually no extra cost to the consumer, and the benefits of continual use are impressive.

For the Expert grower and those with sound understanding of plants physiology

Proteins for transport of water and mineral nutrients across the membranes of plant cells

The uptake and transport of water and mineral ions are among the oldest subjects in plant physiology, and numerous studies have described these processes at the whole-- plant level and at the organ level (e.g., with excised roots). Subsequent work was based on isolated membrane vesicles and electrophysiology to characterize transport processes at the level of the membrane. The recent cloning of the genes for a large number of transport proteins and the availability of knockout mutants make it possible to dissect transport processes in greater detail and to begin to understand the interactions between ion uptake processes that had often been observed at the organ level. This article summarizes recent progress in characterizing the genes and the proteins involved in transporting water and major mineral nutrients.

The plasma membrane of the plant cell is a selectively permeable barrier that ensures the entry of essential ions and metabolites into the cell. Together with the vacuolar membrane (tonoplast), it permits the cytoplasm to maintain intracellular homeostasis. These membranes consist primarily of phospholipid bilayers with transmembrane proteins that permit the traversing of water, ions, and metabolites and the maintenance of a cytosolic pH that is one to three units higher than that of the cell exterior or the vacuole. Pure phospholipid bilayers are permeable to gases, such as 02 and C02, but they are barely permeable to water and nearly impermeable to inorganic ions and other hydrophilic solutes, such as sucrose and amino acids. Proteins are required to transport protons, inorganic ions, and organic solutes across the plasma membrane and the tonoplast at rates sufficient to meet the needs of the cell.

Membranes contain different types of transport proteins: ATPases or ATP-powered pumps, channel proteins, and cotransporters (for other reviews on this topic, see Lalonde et al., 1999; Sze et al., 1999, in this issue). Some of these proteins are well characterized, and their subcellular location is known. The cDNAs for others have been cloned, but functional, biochemical, and cytological data are lacking. ATPases utilize the energy released upon hydrolysis of ATP to move ions across the plasma membrane against chemical and electrical gradients. In plant cells, H^sup +^-ATPases pump protons across the plasma membrane or tonoplast to acidify the extracellular matrix or the vacuole, respectively (Sze et al.,1999). Channel proteins facilitate the diffusion of water and ions down energetically favorable gradients. These proteins form channels through which ions or water molecules pass in single file at very rapid rates-up to 10^sup 6^ to 10^sup 7^ per sec per channel. The activities of channels can be regulated: they can be opened and closed. In plant cells, both water and ion channels have been studied in detail.

Cotransporters, the third class of membrane-transport proteins, can move solutes either up or down gradients at rates of 10^sup 2^ to 10^sup 4^ molecules per second. However, depending on the conditions under which they are assayed, transporters sometimes behave like channels, and vice versa, so that the distinction between them is no longer clear. Classical models assumed that ion channels and cotransporters are clearly distinct proteins. However, recent single-channel studies on animal transporters have demonstrated that cotransporters can also function as ion channels (Fairman et al.,1995; Cammack and Schwartz,1996). Therefore, animal researchers no longer distinguish strictly between ion channels and cotransporters, and models explaining channel modes of transporters have been derived (Larsson et al., 1996; Su et al., 1996). In plant systems, analysis of the wheat HKT1 transporter has provided evidence for two modes of transport by the same protein (Rubio et al., 1995; Gassmann et al.,1996).

Among the cotransporters, we distinguish uniporters, antiporters, and symporters. Uniporters, like channels, move substances down a concentration gradient, albeit much more slowly. Antiporters, also called exchangers, and symporters can move a given substance against its own concentration gradient, the energy requirement for which comes not from ATP but rather is satisfied by an electric potential and/or chemical gradient of a secondary substance. The movement of a solute against its concentration gradient is thus coupled to the movement of the secondary substance-in plants this is often a proton-down its concentration gradient. Thus, the high proton concentration in the apoplast powers the inward movement of certain ions, such as nitrate, by a symporter. Proton gradients can similarly power the movement of ions into the vacuole by the proton-- sodium antiporter or the proton-calcium antiporter.

The ionic compositions of the solutions on either side of both plasma membrane and tonoplast are very different. Not only can the proton concentration be up to 1000 times greater in the apoplast and vacuole than in the cytosol, but gradients of calcium concentration can vary over an even wider range (the concentration of free Ca^sup 2+^ is only 100 nM in the cytosol). The potassium concentration, on the other hand, is much higher in the cytoplasm than in the apoplast. These concentration gradients are maintained by the combined actions of channels, cotransporters, and pumps in the plasma membrane and tonoplast. In addition, at the neutral pH of the cytoplasm, the proteins in the protein-rich cytosolic soup are negatively charged. These and other charge imbalances result in the establishment of an electric potential of -80 to -180 mV at the plasma membrane (negative inside). This potential creates a very strong electric field that provides the energy for biochemical processes at the plasma membrane, such as the uptake of ions or solutes against their concentration gradient, and the opening and closing of ion channels (so-called voltage gated channels).

Many signals coming from the cell exterior (e.g., hormones) can change the electric potential across membranes by activating the H^sup +^-ATPase or opening ion channels. Such a change may only be transient, but it sets in motion a signal transduction cascade that often leads to the activation of specific proteins and genes. Ion channels play important roles in signal transduction, but in this review, we concentrate on the proteins that are involved in nutrient acquisition and water flow.

The Tonoplast and the Plasma Membrane Contain Water Channel Proteins Called Aquaporins

Water movement across cellular membranes is determined not only by osmotic and hydrostatic pressure gradients but also by the intrinsic permeability of the membrane. It was long thought that water molecules could simply diffuse through the lipid bilayer and that variations in water permeability arose from differences in the lipid composition of membranes. This view had to be abandoned with the discovery that proteins, now called aquaporins, specifically facilitate the passage of water through membranes. Such proteins have been found in plants, animals, and bacteria and are probably present in most cellular organisms. In plants, they are found as intrinsic proteins in the tonoplast and the plasma membrane (reviewed in Chrispeels and Maurel, 1994; Maurel, 1997; Schaffner, 1998).

Aquaporins are abundant proteins that may account for 5 to 10% of the total protein in a membrane. Indeed, when intrinsic membrane proteins are subjected to SDS-PAGE, the aquaporins, with molecular masses of 27 to 30 kD, usually form the most abundant polypeptide band. Conventional protein purification methods enable the purification of substantial quantities of aquaporins, provided that a ready supply of membranes is available (Johnson et al., 1989; Johansson et al., 1996). Aquaporins are generally highly antigenic, so that antibodies prepared to total membrane fractions sometimes recognize aquaporins specifically. Proteins other than aquaporins may also form water channels and permit the passage of water through membranes.

Aquaporins are members of the major intrinsic protein (MIP) family. All members of this family are characterized by the presence of six membrane-spanning domains and the signature sequence Asn-Pro-Ala (NPA). This NPA motif is present twice, once in a loop between the second and third membrane-spanning domains and once in a loop between the fifth and sixth membrane-spanning domains. These two loops are thought to be involved in forming the aqueous pore through which water moves. A model of aquaporin structure is presented in Figure 1.

All members of the MIP family are not aquaporins. Some members, such as the GIpF protein of Escherichia coli, facilitate the passage of glycerol, whereas other members transport urea. Some mammalian MIPs have a dual function, permitting the passage of small solutes, such as glycerol, and water, but there is as yet no published evidence on dual-- function aquaporins in plants. Nevertheless, Hertel and Steudle (1997) recently observed that small organic solutes (i.e., alcohols) can slip through the water channels of Chara corallina, and they concluded that water channels may not be as selective as generally believed. The challenge will be to identify the physiological substrates of dual-function aquaporins, because even if plant aquaporins are found to transport glycerol, this may not be their physiological substrate.

All aquaporins are not equally effective at permitting the passage of water across membranes. By surveying the Arabidopsis expressed sequence tag database, we found 23 different expressed MIPs (Weig et al., 1997). Quite a few of these have been verified as aquaporins by expressing the proteins in Xenopus oocytes and measuring the resulting increase in hydraulic conductivity of the oocyte plasma membrane. We do not know if these 23 members are all aquaporins or if some of them have other functions or dual functions. Amino acid sequence comparisons show that the Arabidopsis MIPs form three major groups-one group of tonoplast proteins (TIPs) and two groups of plasma membrane proteins (PIP1 and PIP2)-with a few additional aquaporins unrelated to these three groups.

The three-dimensional structure of mammalian aquaporins has been established from x-ray scattering data of aquaporin crystals. These proteins form tetramers with a central depression between the subunits. The two loops with the NPA motifs are oriented toward a central depression in the tetramer, and the loops of the four subunits probably interact.

Membranes that have abundant aquaporins have a low activation energy for water movement, ~16 kJ mol^sup -1^, which is similar to the movement of bulk water. Water molecules are thought to pass through the aqueous pore in a single file, and an osmotic difference of 100 mM can permit the passage of ~10^sup 6^ water molecules per sec per pore. Within the channels, water molecules experience surroundings similar to those in bulk water.

In spite of the tetrameric structure of aquaporins, the aqueous channel does not form from among the four subunits, but rather each 27-kD polypeptide is a water-conducting unit. For those mammalian aquaporins that transport small solutes, it is possible that the passage of the solute molecules occurs through the center of the tetramer and results from a change in the way the NPA loops interact with each other (Echevarria et al., 1996). Recent evidence shows that two amino acid mutations in the sixth transmembrane domain of an insect aquaporin converts it from a water channel to a glycerol channel (Lagree et al., 1999).

Are aquaporins always "open," or is their activity somehow regulated? Two groups have compared the hydraulic permeability of isolated tonoplast and plasma membrane vesicles and found the tonoplast vesicles to be 10 to 50 times more permeable to water than the plasma membrane vesicles (Maurel et al., 1997b; Niemietz and Tyerman, 1997). Furthermore, the measured activation energy for water movement across the two types of membranes indicated that water movement through the tonoplast vesicles probably involved aquaporins (low energy of activation), whereas water movement across the plasma membrane probably occurred by diffusion through the lipid bilayer (high energy of activation). This rather surprising result indicates that the particular plasma membrane vesicles used (from tobacco cells and wheat roots) had either few aquaporins or aquaporins that were largely inactive.

Recent results (Maurel et al., 1997b; Johansson et al., 1998) indicate that aquaporin activity may be regulated by phosphorylation. Aquaporins have conserved phosphorylation motifs in which certain serine residues are phosphorylated in vivo (Johnson and Chrispeels, 1992; Johansson et al., 1996). Phosphorylation increases the activity of tonoplast aquaporin alpha-TIP from bean and plasma membrane aquaporin PM28A from spinach. This conclusion is based not only on the expression of wild-type and mutant (serine to alanine) aquaporins in Xenopus oocytes but also on the determination of oocyte swelling rates in the presence of inhibitors of protein kinases and protein phosphatases. Interestingly, PM28A is poorly phosphorylated under water deficit, leading to the suggestion that plant cells can at least partially close their aquaporins upon a loss of turgor. Johansson et al. (1998) proposed a model in which loss of turgor caused by water deficit results in the dephosphorylation of PM28A so as to decrease aquaporin activity and conserve water.

With so many aquaporins and/or MIPs, it is not surprising that the corresponding genes are differentially regulated. Through promoter-Beta glucuronidase (GUS) fusion analyses and in situ hybridization with gene-specific probes, the transcription of a number of aquaporin genes has been investigated. These studies show dramatic differences in aquaporin gene expression among various tissues and cell types, but whether differences in transcription correlate with aquaporin levels is unclear. Nevertheless, these gene expression or, more correctly, mRNA abundance studies have led to the following conclusions: (1) many aquaporins are highly expressed in vascular tissues; (2) some tonoplast aquaporins are highly expressed in meristems (where vacuolar biogenesis takes place); and (3) aquaporins are highly expressed in tissues that can experience high water or metabolite flux (see Barrieu et al., 1998, and references therein).

The differential expression of aquaporin genes has been postulated to account for three aspects of water balance in plants. First, the high expression of tonoplast aquaporins in meristematic cells is necessary to sustain vacuole biogenesis and to prepare the cells for the rapid increase in vacuolar volume that accompanies cell enlargement (for a review of vacuole biogenesis, see Marty, 1999, in this issue). Second, in tissues or cells that experience high metabolite fluxes, rapid osmotic equilibration of the cytosol with water from the vacuole may be a prerequisite. Third, in tissues that experience transcellular water flow, aquaporins would greatly reduce the resistance to this flow. Furthermore, because the permeability of the tonoplast is much greater than that of the plasma membrane (Maurel et al., 1997b; Niemietz and Tyerman, 1997), the regulation of this flow may well occur at the plasma membrane.

Much of the evidence for the function of plant aquaporins has been gathered in heterologous systems. What do we know about plant aquaporins in vivo? Maggio and Joly (1995) found that mercuric chloride rapidly reduces pressure-induced root water flux in decapitated tomato plants and that this inhibition is largely reversed by subsequent treatment with Beta-mercaptoethanol. Similarly, many aquaporins are also reversibly inhibited by mercuric chloride, which led the authors to conclude that aquaporins largely account for the hydraulic conductivity of the root system. Tazawa et al. (1996) made similar observations with Chara cells. Barone et al. (1997) recently showed that mercuric chloride induces conformational changes in plant MIPs, and this observation may provide an explanation for the observed inhibition of water transport. However, the possibility that mercuric chloride and Beta-mercaptoethanol exert their effects through other membrane proteins or cellular processes cannot be excluded.

Using a cell pressure probe, Hertel and Steudle (1997) measured the hydraulic conductivity, solute permeability, and reflection coefficients of C. corallina internodes in the temperature range of 10 to 35 deg C. They found that water does indeed use water channels to cross the membrane and that the changes in the reflection coefficient with temperature are a sensitive measure for the open/closed state of the channels. When the channels are open, the energy of activation is ~10 to 15 kJ mol^sup -1^, but when they are closed, the energy of activation increases to 40 to 60 kJ mol^sup -1^.

Transgenic plants demonstrate that water channels are important both at the cellular and at the whole-plant level. Kaldenhoff et al. (1995, 1998) expressed an Arabidopsis IP1b construct in the antisense orientation. The antisense plants had reduced steady-state levels of PIP1a and PIP1b mRNAs, and there was no detectable cross-reactivity with antibodies to PIP1a protein. The protoplasts of the transgenic plants, furthermore, swelled and burst more slowly when exposed to hypotonic conditions than did those of the control plants. Correspondingly, the osmotic water permeability coefficient of the cells was reduced threefold in the antisense plants. Interestingly, the root mass of the antisense plants was five times greater than that of the control plants, although both lines had similar shoot masses and developed similarly. The authors suggested that the reduced water permeability of the plasma membranes caused the plants to overproduce roots as a compensatory mechanism to maintain water flow in the xylem. These findings argue that water flux is generally regulated by the plasma membrane rather than by the tonoplast.

Nitrogen, phosphorus, and sulfur are macronutrients that are required for plant growth and reproduction. These elements are present in the soil solution as organic and inorganic forms, including the anionic species nitrate, phosphate, and sulfate. The uptake of these anions presents a challenge to plants because their concentration in the soil can vary by two to three orders of magnitude and because their uptake often occurs against concentration and electrical gradients. To meet these challenges, plants have evolved active transport systems that are regulated in response to changes in environmental and internal conditions.

The physiological analysis of ion uptake by plant roots has revealed several distinct classes of regulated transport activities. The original work of Epstein classified these activities into two mechanisms (reviewed in Epstein, 1966, 1972). Mechanism I operates at concentrations below 200 (mu)M and behaves as a saturable carrier. At higher concentrations, mechanism II becomes apparent and displays either linear or multiple saturation kinetics. These mechanisms have also been called high- and low-affinity uptake systems, respectively. Figure 2 shows a graphical representation of these types of kinetics.

For anion uptake, both high- and low-affinity systems have a characteristic feature: they are electrogenic. When plant cells are exposed to nitrate, sulfate, or phosphate, an initial depolarization of the membrane is induced, rendering the inside of the cell more positive (for examples, see Ullrich and Novacky, 1990; Glass et al., 1992; Meharg and Blatt, 1995). Depolarization is followed by repolarization of the membrane, which has been attributed to enhanced H^sup +^-ATPase activity in the plasma membrane. The amplitude of the initial depolarization is inversely proportional to the pH of the bath solution and is associated with an alkalization of the extracellular environment. These data indicate that uptake of anions requires cotransport of two or more protons for each anion, although hydroxide antiport cannot be ruled out.

Another characteristic feature of anion uptake is that it is regulated (reviewed in Clarkson and Luttge, 1991; von Wiren et al., 1997; Crawford and Glass, 1998; Schachtman et al., 1998). In the case of phosphate and sulfate, high-affinity transporter activities are derepressed when the plants are deprived of the nutrient. In the case of nitrate, high-affinity transporters are induced by nitrate and are feedback inhibited by reduced and organic forms of nitrogen. These responses allow plants to adjust anion uptake to environmental conditions and internal metabolism.

The physiological studies described above have laid the groundwork for the molecular and genetic analysis of anion uptake (reviewed in Tanner and Caspari,1996; von Wiren et al., 1997; Crawford and Glass, 1998; Forde and Clarkson, 1998; Schachtman et al.,1998). A major challenge has been to identify the components of each uptake system and determine the contribution that each makes to nutrient uptake and plant growth. Many anion transporter genes have now been cloned, and all have predicted membrane topologies found in cotransporters with 12 membrane-spanning domains. Many of the cloned genes also show regulated expression and produce functional products in heterologous systems such as yeast or Xenopus oocytes. In one case, plants with mutations in the transporter gene have been identified and used for functional analysis. The initial conclusions from this work are that multiple, regulated genes are involved in the uptake of a particular anion and that plant transporters share sequence and functional similarity with equivalent proteins in fungi and yeast (see below).

In most soils, phosphate is not mobile and is quickly depleted from the rhizosphere. Often, plants must take up soluble, inorganic phosphate from concentrations of <10 (mu)M while maintaining cytosolic concentrations of 5 to 10 (mu)M (Lee and Ratcliffe, 1993). Plant roots have a high-affinity uptake system that is specifically derepressed by phosphate deprivation (reviewed in Clarkson and Luttge, 1991; Schachtman et al., 1998). The cloning and analysis of plant phosphate transporter genes were made possible by the availability of fungal and yeast transporter mutants and clones. The plant genes show extensive sequence similarity among themselves, and they show regulation in response to phosphate starvation (reviewed in Schachtman et al., 1998).

Eukaryotic high-affinity phosphate transporter genes were initially isolated from Saccharomyces cerevisiae (PH084; Bun-ya et al., 1991), Neurospora crassa (PHO-5; Versaw, 1995), and the arbuscular mycorrhizal fungus Glomus versiforme (GvPT; Harrison and van Buuren, 1995). cDNA clones with related sequences were then identified in the Arabidopsis expressed sequence tag databases. Subsequently, phosphate transporter genes were isolated from Arabidopsis (Muchhal et al., 1996; Lu et al., 1997; Smith et al., 1997a), tomato (Daram et al., 1998; C. Liu et al., 1998), potato (Leggewie et al., 1997), and Medicago truncatula (H. Liu et al., 1998). The plant genes typically show 75 to 85% identity among themselves and 25 to 35% identity with the fungal transporters at the amino acid level. In one case, two genes (APT1 and APT2) are 99% identical and are genetically linked (Smith et al., 1997a). All of the plant genes respond to phosphate deprivation with a dramatic increase in transcript levels, and most of the corresponding proteins are localized in the roots. For the tomato genes, expression is primarily restricted to the root epidermis (C. Liu et al., 1998). Interestingly, both M. truncatula genes are downregulated when roots are colonized by mycorrhizal fungi (H. Liu et al., 1998). Lastly, the functional analysis of the plant phosphate transporters showed that most complement the yeast pho84 high-affinity phosphate transporter mutant (Bun-ya et al., 1991).

The findings summarized above indicate that the cloned plant genes encode high-affinity phosphate transporters. One would therefore expect that these transporters would have a K^sub m^ between 1 and 10 (mu)M for phosphate uptake. However, the measured K^sub m^ values for the plant transporters expressed in yeast are all above 30 (mu)M: 130 (mu)M for StPT2 and 280 (mu)M for StPT1 from potato,192 (mu)M for MtPT1 from M. truncatula, and 31 (mu)M for LePT1 from tomato (Leggewie et al., 1997; Daram et al., 1998; H. Liu et al., 1998). To explain this discrepancy, a recent report suggests that the high K^sub m^ values observed in yeast are due to the foreign environment in which these plant proteins are expressed (Mitsukawa et al., 1997). This work found enhanced phosphate uptake activity in tobacco tissue culture cells that overexpress the APT2 transporter (also named AtPT1 and PHT1) even though no APT2 complementation of the yeast pho84 mutant was detected. The phosphate uptake activity in tobacco attributed to the APT2 gene had a K^sub m^ of 3 (mu)M and was inhibited by protonophores, indicating that APT2 is a high-affinity proton/phosphate symporter. To explain the difference in activity or K^sub m^ values between yeast and plants, it has been suggested that the plant phosphate transporters may be modified in plants. Alternatively, they may interact with other plant proteins (as has been postulated in yeast [Bun-ya et al., 1996]) to generate a transport complex with altered kinetic properties (Mitsukawa et al., 1997; Smith et al..1997a).

The fungal genes described above are all H^sup +^-phosphate symporters; however, fungi also possess Na^sup +^-phosphate symporters. A genome database search shows that a homolog to the Na^sup +^-phosphate symporter PHO-4 exists in the Arabidopsis genome (GenBank accession number X97484), indicating that Na+ coupling may also exist in higher plants (see below for coupling to K^sup +^).

Identification of Sulfate Transporters with Different Affinities for Sulfate and Different Expression Patterns

Sulfate uptake is characterized by a starvation-induced, high-affinity system with a K^sub m^ between 10 and 20 (mu)M along with a nonsaturable, low-affinity system (Legget and Epstein, 1956; reviewed in Clarkson and Luttge, 1991; Hawkesford et al., 1993). High-affinity sulfate transporter genes were originally identified in N. crassa (Ketter et al., 1991) and yeast (Smith et al., 1995b). A yeast mutant was used to clone three sulfate transporter cDNA clones from the tropical legume Stylosanthes hamata (Smith et al., 1995a). The SHST1 and SHST2 proteins are 95% identical and, when expressed in yeast, show an acid-enhanced uptake of sulfate with a K^sub m^ of 10 (mu)M. Expression of the SHST1 and SHST2 genes is root specific and derepressed by sulfate deprivation. The third gene, SHST3, belongs to a different class because it is ~50% identical to the first two genes (at the amino acid level), displays a higher K^sub m^ (100 (mu)M) for sulfate uptake in yeast, and expresses much less mRNA in plants than do the SHST1 and SHST2 genes. The SHST3 gene has another interesting property: its mRNA levels are higher in shoots than in roots of plants supplied with adequate sulfur but may decrease (in the leaves) under sulfate limiting conditions.

Additional sulfate transporter genes have more recently been identified in Arabidopsis (AST56 and AST68; Takahashi et al., 1996, 1997) and barley (HVST1; Smith et al., 1997b). The HVST1 protein is most similar in sequence and activity to the SHST1 and SHST2 proteins of S. hamata. HVST1 expressed in yeast shows a K^sub m^ of 7 (mu)M for sulfate uptake, and its mRNA is found exclusively in roots, increasing in abundance in response to sulfate deprivation. The products from the two Arabidopsis genes, however, are more similar to SHST3 (60% peptide sequence identity) than to the SHST1 and SHST2 proteins (50% identity). AST56 and AST68 expression patterns are also not root specific. AST56 is constitutively expressed, whereas AST68 is induced with sulfate starvation.

These findings indicate that the SHST1/SHST2/HVST1 class of sulfate transporters is part of the starvation-- induced, high-affinity uptake system in plant roots. The second class (composed of SHST3, AST56, and AST68) is thought to contain low-affinity transporters that either load sulfate into the vascular tissue in roots and unload sulfate into leaf cells (for AST68; Takahashi et al., 1997) or transport sulfate internally between cellular or subcellular compartments (for SHST3; Smith et al., 1995a). No transporters have been assigned to a low-affinity sulfate uptake system in the root.

Inorganic nitrogen nutrition differs from that of phosphate and sulfate in that not one but two inorganic forms, ammonium and nitrate, are readily used by plants (in addition, N^sub 2^ gas is used by nitrogen fixers). For many plants, the presence of both ammonium and nitrate is optimal for growth, but nitrate is taken up in the largest quantity. High-affinity nitrate uptake is characterized by K^sub m^ values for nitrate between 7 and 80 (mu)M and saturation below 200 (Mu)M (reviewed in Glass and Siddiqi, 1995; Crawford and Glass, 1998). High-affinity uptake activity also displays both positive and negative regulation. In plants that have not been exposed to nitrate, high-affinity uptake activity is detected at a basal level. When a root is exposed to nitrate, high-affinity uptake activity increases, and a new activity with a distinct K^sub m^ appears. If nitrate accumulates in the root or if the root is exposed to ammonium or certain amino acids, uptake is downregulated. Plants also possess another uptake activity that is detectable at nitrate concentrations above 0.2 to 0.5 mM and that displays linear (nonsaturable) kinetics. This low-affinity activity does not appear to be induced by nitrate in plants such as barley. From these findings, a model was proposed that plants have three nitrate uptake systems: a constitutive high-affinity uptake system (cHATS), an inducible high-affinity system (iHATS), and a constitutive low-- affinity system (cLATS) (reviewed in Glass and Siddiqi, 1995; Crawford and Glass, 1998).

Efforts to identify the components of these three uptake systems led to the cloning and characterization of several nitrate transporter genes (reviewed in Crawford and Glass, 1998). Two gene families, named NRT1 and NRT2, have been identified so far. The first gene identified in the NRT2 family was crnA of Aspergillus nidulans (Unkles et al., 1991). A mutation in this gene reduces nitrate uptake two- to fourfold in conidiospores and young mycelia but not in older mycelia (Brownlee and Arst, 1983). Subsequently, a pair of crnA-related algal genes (Nrt2;1 and Nrt2;2, originally called nar-3 and nar-4) was isolated from Chlamydomonas (Quesada et al., 1994). Mutations in these genes impair high-affinity nitrate uptake. Some time later, NRT2 genes were identified in higher plants (reviewed in Crawford and Glass, 1998). The expression of these genes is root specific, nitrate inducible, and ammonium/glutamine repressible. Recently, an NRT2 gene (YNT1) was isolated from the yeast Hansenula polymorpha, and a mutant defective in nitrate uptake was generated by disrupting the YNT1 gene (Perez et al.,1997). A barley NRT2 cDNA, under the control of a suitable yeast promoter, has been shown to partially restore high-affinity nitrate uptake in the yntl mutant (B. Forde and J. Siverio, personal communication). These data indicate that the NRT2 genes are components of the iHATS in plants.

The first member of the NRT1 family was identified in a chlorate-resistant mutant (chl1) of Arabidopsis that had reduced nitrate uptake above 1 mM nitrate (i.e., in the low-- affinity range) (Doddema and Telkamp, 1979; Tsay et al., 1993). The CHL1 gene was cloned from a T-DNA insertion mutant and shown to encode a protein with electrogenic nitrate uptake activity in Xenopus oocytes (Tsay et al., 1993). Initial experiments showed that CHL1 has a K^sub m^ of ~8 mM for nitrate in Xenopus oocytes (Huang et al.,1996). Figure 3 is representative of the data showing the depolarization response to nitrate in Xenopus oocytes expressing CHL1. In plants, the CHL1 gene is nitrate induced, has higher mRNA levels at acidic pH, and is expressed predominantly in the outer layers of the root (i.e., endodermis to epidermis) after nitrate induction (Tsay et al., 1993; Huang et al., 1996). Together with physiological data showing that chl1 mutants are defective in low-affinity nitrate uptake (Doddema and Telkamp,1979), these findings indicated that CHL1 is a component of the low-affinity nitrate uptake system.

The assignment of CHL1 to the low-affinity system best fits the physiological data in Arabidopsis; however, it ran counter to previous descriptions of the low-affinity system in other plants, that is, cLATS shows no sign of nitrate induction. A solution to this contradiction became apparent when it was discovered that the contribution of CHL1 to low-affinity nitrate uptake is variable and depends on the composition of the nutrient solution and that more than one differentially regulated NRT1 gene exists in plants (Huang et al., 1996; Touraine and Glass, 1997). If Arabidopsis plants are grown with nitrate but no ammonium, short-term, low-affinity nitrate uptake rates for wild-type and chl1 mutant plants are similar (12 (mu)mol g^sup -1^ hr^sup -1^ at 5 mM nitrate); however, if plants are grown on ammonium nitrate, uptake rates are much lower for chl1 mutants (2 (mu)mol g^sup -1^ hr^sup -1^ at 5 mM nitrate) than for the wild type (7.5 (mu)mol g^sup -1^ hr^sup -1^) (Touraine and Glass, 1997). The implication from these results is that the contribution that CHL1 makes to low-affinity uptake depends on the relative expression or activity levels of two transporters. A candidate gene that could encode this second transporter has been identified in Arabidopsis and is called NRT3 (Huang et al., 1996; see also Genbank accession number AF073361). Interestingly, this protein is a member of the NRT1 family, being 36% identical to CHL1.

The same situation may also apply in tomato, in which two NRT1 genes that are 65% identical to CHL1 have been cloned (Lauter et al.,1996). The first is expressed predominantly in root hairs and is nitrate inducible. The second is more ubiquitously expressed in roots and is not nitrate regulated. Given their high sequence identity to CHL1 and their expression in roots, it is possible that both genes encode nitrate transporters that are components of the low-affinity system.

The results described above appeared to fit into a simple model: members of the NRT1 gene family, which includes CHL1, encode the transporters of the low-affinity system (which has both constitutive and inducible components); and NRT2 genes make up the inducible high-affinity system. Our understanding of these systems took an interesting turn, however, when a high-affinity nitrate uptake mutant was discovered in Arabidopsis that had a transposon insertion in the CHL1 gene (R. Wang et al.,1998). Further analysis demonstrated that CHL1 is a major component of the high-affinity uptake system when plants are grown on ammonium nitrate but that it plays a minor role when ammonium is absent Touraine and Glass, 1997; R. Wang et al., 1998; Liu et al.,1999). Thus, as is the case in the LATS, the contribution of CHL1 depends on the composition of the nutrient solution.

Further work showed that CHL1 contributes to both iHATS and cHATS (R. Wang et al., 1998). Its contribution to the constitutive system is pH dependent, being most significant at acidic pH. To explain how CHL1 could be a major component of both LATS and HATS, it was proposed that CHL1 is a dual-affinity transporter with both high- and low-affinity K^sub m^ values (R. Wang et al., 1998; Liu et al., 1999). These new findings show that nitrate uptake is not mediated by a one-- affinity-type/one-transporter system but is more complex and dynamic, with multiple transporters contributing to a given system (e.g., at least two NRT1 genes for LATS) and a single transporter contributing to multiple uptake systems in an environmentally dependent manner (e.g., CHL1 contributes to cHATS, iHATS, cLATS, and iLATS when ammonium is present and/or the pH is low).

Positively charged macronutrients such as potassium (K+), ammonium (NH^sub 4^^sup -^), calcium (Ca^sup 2-^), and magnesium (Mg^sup 2+^) are required in relatively large amounts for plant growth and development. Additional cationic micronutrients (iron, manganese, zinc, copper, and nickel) play essential roles as cofactors and activators of enzymes. In the past few years, transporters for several of these cations have been cloned from plants, including transporters for ammonium, iron, copper, and potassium (Sentenac et al., 1992; Ninnemann et al., 1994; Kampfenkel et al., 1995; Eide et al., 1996). As is the case for H^sup +^-ATPases (Sussman, 1994), data from many laboratories show that for each cation, multiple genes and even multiple gene families appear to be responsible for transport. This is not surprising, considering that different plant tissues have different nutritional and energy requirements. Furthermore, the transport of nutrients across a variety of different membranes (e.g., plasma membrane, tonoplast, and the inner and outer plastid membranes) is required. In addition, multiple membrane proteins may be needed for cation uptake from soils to adapt to varying extracellular conditions and nutrient availability. To illustrate the underlying complexity and open questions, we focus here on recent advances in the understanding of K^sup +^ transport mechanisms.

Potassium is the most abundant cation in plants and plays an important role in processes such as cell elongation, leaf movements, tropisms, metabolic homeostasis, germination, osmoregulation, stomatal movements, and sodium (Na^sup +^) stress (reviewed in Kochian and Lucas, 1988; Maathuis et al., 1997). Because no convenient radioactive isotope of K^sup +^ exists, transport of K^sup +^ is measured with radioactive Rb^sup +^. Classical studies of K^sup +^ (Rb^sup +^) uptake into roots showed two main transport components, described as high-affinity (mechanism I) and low-affinity (mechanism II) transport, respectively (reviewed in Epstein, 1972; see Figure 2). One of the high-affinity K^sup +^ uptake components is induced by removing K^sup +^ from the nutrient medium.

A comparison of K^sup +^ uptake in K^sup +^-starved and K^sup +^-supplied barley and maize roots shows constitutive and inducible high-affinity K^sup +^ uptake components (Glass and Dunlop, 1978; Kochian and Lucas, 1982). The genetic separation of constitutive from K^sup +^ starvation-induced high-affinity K^sup +^ uptake systems was recently demonstrated with the sodium-sensitive Arabidopsis mutant sos1 (Wu et al., 1996). Specifically, uptake studies showed that the sos1 mutant is defective in inducible high-affinity K^sup +^ uptake, whereas constitutive (i.e., background) high-affinity K^sup +^ unaltered (Ding and Zhu, 1997). Because of the importance of K^sup +^ uptake for plant nutrition, research in several laboratories is focused on determining the transport mechanisms and molecular bases of high- and low-affinity K^sup +^ transport components.

Multidisciplinary studies have been pursued to determine the nature of the proteins (e.g., ATPase, cotransporter, and/ or channel) that might mediate high- and low-affinity K^sup +^ uptake in roots. In intact maize roots, the nonsaturable low-- affinity component of Rb+ uptake is inhibited by the K^sup +^ channel blocker tetraethylammonium, suggesting the involvement of K^sup +^channels in low-affinity K^sup +^ (Rb^sup +^) uptake (Kochian and Lucas, 1982). Patch clamp studies of different root cell types (root cortex, root hair, stele, and parenchyma) in several laboratories demonstrated the existence of hyperpolarization-activated K^sup +^ (K^sup +^^sub in^) channels, leading to the suggestion that these K^sup +^^sub in^ channels may contribute to low-- affinity channel-mediated K^sup +^ uptake (reviewed in Maathuis et al., 1997). These K^sup +^ channels are activated by plasma membrane potentials more negative than the K^sup +^ equilibrium potential, suggesting that activated K^sup +^^sub in^ channels "draw" K^sup +^ into cells upon sufficient activation of proton pumps. Tracer flux studies suggest that multiple low-affinity K^sup +^ (Rb^sup +^) uptake components exist, with distinguishable K^sub m^ values for K^sup +^ in the millimolar concentration range (Epstein, 1972). Further research is needed to determine the relative contributions of K^sup +^^sub in^ channels in different root cells to physiological K^sup +^ uptake.

A Single Mechanism for High-Affinity K^sup +^ Transport Could Not Be Resolved by Studies with Intact Roots

Because of the importance of high-affinity K^sup +^ uptake for plant growth, the molecular bases and transport mechanisms used to accumulate K^sup +^ from micromolar K^sup +^ concentrations are of particular interest. In N. crassa, H^sup +^-K^sup +^ symport activity is responsible for high-affinity K^sup +^ uptake (Rodriguez-Navarro et al., 1986). The physiological mechanisms for high-affinity K^sup +^ nutrition in plants have, however, remained more elusive. For example, Kochian, Lucas, and colleagues pursued elegant characterizations of high-affinity K^sup +^ uptake in maize roots using tracers, intracellular K^sup +^ and pH electrodes, extracellular K^sup +^-gradient sensing (vibrating) electrodes, and membrane potential recordings (Newman et al., 1987; Kochian et al., 1989). The K^sub m^ for K^sup +^ uptake in maize roots was 4 to 6 (mu)M and that for Rb^sup +^ uptake was 30 pIM, indicating a higher apparent affinity for K+ than for Rb^sup +^ (Newman et al., 1987). Exposure to extracellular micromolar K^sup +^ concentrations produced a rapid depolarization of the plasma membrane, suggesting that the K^sup +^ uptake mechanism is not electroneutral and that uptake of the positively charged K^sup +^ ions contributes to this depolarization (Newman et al.,1987). Therefore, an electroneutral H^sup +^-K^sup +^ exchange is unlikely to be the mechanism for high-affinity K^sup +^ uptake. Interestingly, however, in these detailed studies with intact roots, no single K^sup +^ transport mechanism could be identified that mediates high-affinity K^sup +^ uptake. For example, no proton stimulation or evidence for proton-K^sup +^ exchange could be obtained with regard to the high-affinity system, and ATP-dependent K^sup +^ uptake pumping also was thought to be unlikely (Newman et al.,1987; Kochian et al.,1989). Studies with barley roots also show no proton stimulation of high-- affinity K^sup +^ uptake (Glass and Siddiqi,1982).

Other nutrients, including ammonium, nitrate, phosphate, and sucrose, show a clear stimulation of nutrient uptake by the physiological proton gradient in intact organs (McClure et al., 1990; Meharg and Blatt, 1995). However, no proton stimulation of K^sup +^ uptake has yet been reported in intact roots. Proton stimulation of high-affinity K^sup +^ uptake was indicated in subtractive current-voltage analyses of Arabidopsis root cells (Maathuis and Sanders,1994), but this activity (-5 to 15 pA per cell) was observed in <=10% of patch clamp experiments (Maathuis and Sanders, 1997), and the relative contribution of this activity to whole-root uptake has not yet been determined. Disruption of genes for other mechanisms of K^sup +^ uptake could provide an approach to resolve the proposed proton-stimulated high-affinity K^sup +^ uptake in roots.

The finding that the wheat HKT1 cDNA encodes a Na^sup +^-K^sup +^ symporter when expressed in yeast and Xenopus ooctyes was surprising. This is because other nutrients in plants are transported via symport with protons rather than Na^sup +^ (Rubio et al.,1995) and because coupling of high-affinity K^sup +^ uptake to Na^sup +^ uptake was not found in wheat roots, where the gene is expressed (Maathuis et al., 1996). At low concentrations, Na^sup -^ is known to stimulate root growth (Flowers and Lauchli, 1983, and references therein), but the mechanisms remain unknown. It is interesting that, in certain aquatic plants (e.g., Charophyte algae, Egeria and Elodea roots, and in Vallisneria and Egeria leaves), Na^sup +^-K^sup +^ symport has been identified as a high-affinity K^sup +^ uptake mechanism (Smith and Walker, 1989; Walker and Sanders, 1991; Maathuis et al., 1996). These data show that Na^sup +^-coupled high-affinity K^sup +^ uptake activity exists in these plants. Whether the underlying transporters are related to HKT genes has not been analyzed. It is possible that in these aquatic plants, a single K^sup +^ transport mechanism could be resolved because one major type of K^sup +^ uptake transporter is responsible for the bulk of high-affinity K^sup +^ uptake, whereas high-affinity K^sup +^ uptake in terrestrial plants has been proposed to require many types of transporters to adapt to environmental and soil conditions (Rubio et al., 1996).

Three gene families have been identified and proposed to contribute to high-affinity K^sup +^ transport in plants, including the Arabidopsis root inward-rectifying K^sup +^ channel AKT1 (Sentenac et al., 1992; Hirsch et al., 1998), the wheat root Na^sup +^-K^sup +^ transporter HKT1 (Schachtman and Schroeder, 1994), and the recently identified gene family of HAK/KUP transporters in barley (HvHAK) and Arabidopsis (AtKUP or AtKp (Quintero and Blatt,1997; Santa-Maria et al.,1997; Fu and Luan, 1998; Kim et al., 1998). Promoter-GUS studies suggest that AKT1 is predominantly expressed in the epidermis, cortex, and endodermis of mature Arabidopsis roots (Lagarde et al.,1996). As seen in Figure 4, mRNA in situ hybridization analyses show HKT1 expression in the cortex of wheat roots and in cells surrounding the vasculature of wheat leaves (Schachtman and Schroeder, 1994). In rice roots, an HKT1 homolog is expressed in the epidermis and endodermis (Golldack et al.,1997).

HKT1 is related to TRK1 and TRK2, the yeast genes encoding plasma membrane high-affinity K^sup +^ uptake transporters (Schachtman and Schroeder,1994). Similarly, the HAK/ KUP genes in barley and Arabidopsis show similarity to the bacterial low-affinity Kup and the Schwanniomyces occidentalis high-affinity HAK1 K^sup +^ uptake transporters (Schleyer and Bakker, 1993; Banuelos et al., 1995). The HAK/KUP transporters represent a large gene family in Arabidopsis (Quintero and Blatt,1997; Kim et al.,1998), and genome database searches indicate that this species possesses at least 10 members. The transport mechanisms of the HAK/ KUP transporter family members remain uncharacterized.

Expression of the Arabidopsis HAK/KUP homolog AtKUP2 in yeast results in low-affinity Rb^sup +^ uptake (Quintero and Blatt, 1997). Expression of the barley homolog HvHAK in yeast and expression of an Arabidopsis homolog AtKUP1 in Arabidopsis suspension cells results in high-affinity Rb^sup +^ uptake (Santa-Maria et al.,1997; Kim et al.,1998). In addition, AtKUP1 can show both high- and low-affinity Rb+ uptake activity, as discussed below (Fu and Luan,1998; Kim et al., 1998). Interestingly, HvHAK and AtKUP3 mRNA levels increase markedly in barley and Arabidopsis roots, respectively, after K^sup +^ starvation, indicating a putative contribution of HAK/KUP gene products to inducible high-affinity K^sup +^ uptake (Santa-Maria et al., 1997; Kim et al., 1998). In barley, wheat, and rice, HKT1 mRNA levels are also enhanced in response to K^sup +^ withdrawal, with rapid induction kinetics (Golldack et al.,1997; T.-B. Wang et al.,1998). It is therefore possible that HvHAK, AtKUP3, and HKT1 contribute to K^sup +^ starvation-induced K^sup +^ transport in the analyzed plant species.

How are the activities of K+ transport proteins regulated? In addition to transcriptional regulation, post-translational regulation seems likely to play an important role in K^sup +^ uptake. Several mechanisms for post-translational regulation of K^sup +^^sub in^ channels in guard cells and other plant cells have indeed been reported (reviewed in Muller-Rober et al., 1998). In addition, genetic studies of sos mutants in Arabidopsis indicate that single genetic loci may regulate multiple K^sup +^ and Na^sup +^ transport components in parallel, suggesting that central regulation mechanisms exist for K^sup +^ transport (Wu et al., 1996; Liu and Zhu, 1997). The Na^sup +^ sensitivity mutant sos3 regulates K^sup +^ (Rb^sup +^) and Na^sup +^ uptake and was recently shown to encode a homolog of a Ca2^sup +^-sensing regulatory subunit that modulates protein phosphatases or kinases in animal cells (Liu and Zhu, 1998). In fact, ectopic expression of constitutively active mutants of the yeast protein phosphatase calcineurin showed enhanced NaCI tolerance (Pardo et al., 1998). These data demonstrate the importance of posttranslational regulation for K^sup +^ and Na^sup +^ transport. Further biochemical, electrophysiological, and genetic analyses will help to determine the regulation and physiological contributions of individual K^sup +^ transporters in vivo.

Disruption of the Inward K^sup +^ Channel AKT1 Indicates that Single Transporters May Mediate Both Low- and High-- Affinity K^sup +^ Uptake

The classical separation of low- and high-affinity K^sup +^ transport components is being reconsidered based on recent findings. Most strikingly, a loss-of-function mutation of the AKT1 K^sup +^^sub in^ channel gene (akt1-1) has recently been shown to cause reduced growth of Arabidopsis seedlings at micromolar extracellular K^sup +^ concentrations (Hirsch et al., 1998). Moreover, Rb^sup +^ uptake analyses, membrane potential recordings, patch clamp recordings, plant growth assays, and genetic segregation analysis suggest that the AKT1 K ^sup +^ channel, previously thought to constitute a low-affinity K^sup +^ transporter, can in fact function in high-affinity K^sup +^ uptake. Previous theoretical discussions have also led to the suggestion that high- and low-affinity components may be mediated by the same transporter (Nandi et al., 1987) and from guard cell studies that K^sup +^ channels could contribute to high-- affinity K^sup +^ uptake when other transporters are inactivated (Schroeder et al., 1994). Expression of AKT1 in yeast also allows dual-affinity Rb^sup +^ uptake at micromolar concentrations as well as at millimolar K^sup +^ concentrations (Sentenac et al., 1992).

One explanation for a "dual-affinity" nature of single transporters could lie in the fact that extracellular ions, particularly K^sup +^ ions, have a significant influence on the membrane potential of cells. For example, at micromolar extracellular K^sup +^ concentrations, the membrane potential is extremely negative (-230 mV) (Kochian et al., 1989; Hirsch et al., 1998), thus facilitating proton pump-driven K^sup +^ uptake through channels. When other high-affinity K^sup +^ transporters are inactivated or blocked, Kt channels allow high-affinity K^sup +^ uptake (Hirsch et al.,1998). For example, addition of ammonium to the growth medium is necessary to retard growth of the akt-1-1 mutant at micromolar K+ concentrations. NH^sub 4^^sup +^ inhibits Rb^sup +^ uptake in Arabidopsis roots (Hirsch et al., 1998), and it also inhibits Rb^sup +^ uptake by the HvHAK1 transporter (Santa-Maria et al.,1997) and by the AtKUP1, 2, and 3 transporters (E.J. Kim and J.l. Schroeder, unpublished data). Together, these data indicate that repression or NH4+ inhibition of some of the high-affinity K^sup +^ uptake pathways results in conditions whereby AKT1 contributes to growth at micromolar K^sup +^ concentrations (Hirsch et al.,1998). Furthermore, the finding that disruption of the AKT1 gene reduces Arabidopsis growth at micromolar K^sup +^ concentrations in the presence of NH^sub 4^^sup +^ provides a new experimental system in which to further examine hypotheses regarding multiple affinities by an individual transporter in vivo.

Evidence for dual-affinity K^sup +^ uptake by a single transporter was also found for a member of the HAK/KUP transporter family. Expression of the AtKUP1 cDNA in Arabidopsis suspension cells (Kim et al., 1998) or in yeast cells (Fu and Luan, 1998) gives rise to simultaneous high- and low-affinity Rb^sup +^ uptake fluxes.

On the other hand, kinetic studies of HKT1-induced Rb+ and K^sup +^ uptake in yeast show only a saturable high-affinity component for uptake (Rubio et al., 1995; Gassmann et al., 1996). However, for Na^sup +^ ions, the HKT1 transporter has been shown to mediate high-affinity and low-affinity Na^sup +^ uptake in yeast and Xenopus oocytes (Rubio et al., 1995; Gassmann et al., 1996). In this case, the high- and low-affinity Na^sup +^ uptake components can be mechanistically separated: high-- affinity Na^sup +^ uptake occurs via Na^sup +^-K^sup +^ symport, whereas low-affinity Na^sup +^ uptake is possibly more channel-like and does not require cotransport of K^sup +^ (Rubio et al., 1995). Recent studies on K^sup +^ channels, HAK/KUPs, and HKT1 point to an emerging theme in which single transporters may function physiologically over broad substrate concentration ranges depending on growth conditions.

The capacity of HKT1 to function as an Na^sup +^ uptake pathway at millimolar Na^sup +^ concentrations indicates possible roles for HKT1 in physiological Na+ transport and Na^sup +^ toxicity in root and leaf cells (Rubio et al., 1995). Note that Na^sup +^ influx data in roots also show multiple components for low-- affinity Na^sup +^ uptake (Epstein, 1972). As for K^sup +^ uptake, models assuming one major pathway for low-affinity Na^sup +^ uptake are unlikely to survive in the wake of findings of multiple gene families mediating cation uptake.

The physiological reasons for the existence of large gene families for the transport of plant nutrients have not yet been experimentally determined. However, several considerations provide plausible explanations. First, redundancy in the essential mechanisms for nutrient accumulation may be important for the survival of plants. Second, as discussed above, the large physiological variation in soil and apoplastic concentrations of nutrients and toxic competitor ions may call for an array of nutrient transport mechanisms to ensure condition-dependent nutrient uptake. Third, expression in different membranes (e.g., plasma membrane versus tonoplast) and tissues (e.g., root hairs versus phloem) is important and has not yet been analyzed in detail, particularly for K+ transporters. Fourth, constitutive and inducible high-- affinity uptake components can respond to changing nutrient availabilities and ionic conditions. For example, AKT1 expression levels were reported not to be affected by K^sup +^ starvation in Arabidopsis roots (Lagarde et al., 1996). Therefore, AKT1 has been hypothesized to contribute to constitutive K^sup +^ uptake in Arabidopsis (Lagarde et al., 1996). Studies in the past three years and earlier research by Kochian et al. (1989) show that the older models assuming one major pathway for high-affinity K^sup +^ uptake are not adequate to analyze the underlying biological complexity of K^sup +^ nutrition.

Significant advances have been made in characterizing cDNAs and in analyzing the biophysical transport mechanisms of individual plant K+ transporters. Recent findings nevertheless point to many unresolved central questions regarding integrated K+ nutrient uptake in the plant. The inability thus far to resolve individual high-affinity K^sup +^ uptake mechanisms in whole roots of terrestrial plants suggests that the transporter families discussed above, and perhaps others as yet unknown, may function in parallel. Loss-of-- function mutations, such as chl and akt1-1, can provide insight into the contributions of individual genes to nutrient uptake under different growth conditions. Important open questions, including tissue-specific expression, membrane targeting, post-translational regulatory mechanisms, and regulatory responses to differing environmental conditions, are currently being addressed. The ability to disrupt individual genes in plant species such as Arabidopsis and maize, together with interdisciplinary analyses, will provide approaches for determining the roles of individual K- transporters to many essential plant physiological processes.

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