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Cycling silicon – the role of accumulation in plants Silicon, the element in so many plant scientists’ minds as we come into an age of research ‘in silico’, still presents an enigma when it comes to its nutritional role in higher land plants. We should all be well aware of it – the second most abundant element in the Earth’s crust, which occurs in the soil solution at 0.1–0.6 mol m−3 as Si(OH)4 (two orders of magnitude higher than the macronutrient phosphorus occurs as H2PO4–/HPO42– – Epstein, 1999; Datnoff et al., 2001). Yet silicon is not an essential element for any of the embryophytes tested, and the dry matter of these contains very variable amounts of the element – 1.3–47.3 mg per g of plant dry matter (Epstein, 1999; Ma et al., 2001). Essential, in strict plant nutritional terms, means that the plant cannot complete its life cycle, under otherwise optimal conditions, in as near to the absence of the element as techniques will allow. Clearly we need to know more about the role of silicon in plants, and in this issue (pp. 431–436) Tamai & Ma address the most basic of questions – how does the silicon get into the plant in the first place? (see also Lux et al. pp. 437–441 in this issue, who have examined silicon in sorghum). The study species used by Tamai & Ma, rice, is an especially intriguing case because, as is well known, rice is a major silicon accumulator.

What do we know about silicon in plants? While silicon is not essential for the growth of higher plants, we do know that its availability influences many aspects of the biology of plants that naturally have moderate to high levels of the element (Epstein, 1999; Datnoff et al., 2001). Examples are restriction of grazing and parasitism, increased light interception, and alleviation of the effects of deficiency or excess of nutrient and other solutes (Epstein, 1999; Datnoff et al., 2001; Britez et al., 2002). Thus, although silicon is not essential for higher plants it very significantly improves fitness in nature and increases agricultural productivity. Photosynthetic organisms other than higher plants can also have an important involvement with silicon. Of these the most globally significant are the diatoms (Bacillariophyceae: Heterokontophyta), with an absolute requirement. Cell walls, or frustules, of diatoms are silicified – among the roles of these silicified walls is that of mechanical protection from grazers (Hamm et al., 2003).

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The global silicon cycle Plants (in the broad sense) are not only involved with silicon in terms of growth, but are also major components of the global silicon cycle. Taking the land surface as a starting point, vascular plants play a major role in weathering silicate rocks (Berner & Berner, 1996; Lucas, 2001; Raven & Edwards, 2001). ‘Biological pumping’ of CO2 from the bulk atmosphere to the soil atmosphere involves photosynthesis by shoots, translocation of organic carbon to the roots, and respiration by plants and soil biota of living and dead plant material (Lucas, 2001). The restricted diffusion pathway to the bulk atmosphere gives a steady state CO2 concentration in the soil atmosphere which is one or two orders of magnitude higher than that in the bulk atmosphere. The high concentration of CO2 in the soil solution increases the rate at which CO2 reacts with silicate rocks to yield soluble silicic acid and the soluble bicarbonate salts of the metals from the silicates (Berner & Berner, 1996; Lucas, 2001). Most soil solution water ultimately reaches the ocean, providing, with a minor component from the reaction of seawater with basalt, the silicic acid input to the oceans. Silicic acid is removed from the ocean by long-term incorporation into sediments of a small fraction (a few per cent) of the biogenic silica which is precipitated in intracellular compartments after active influx of silicic acid in, predominantly, planktonic diatoms (Berner & Berner, 1996; Falkowski & Raven, 1997; De Master, 2002). Most of the silica produced by diatoms is recycled to silicic acid in the ocean water. The cycle of silicon is completed by reactions at high temperature and pressure in the Earth’s crust. Silica, with sedimented carbonates produced biologically from bicarbonate, is reconverted to metal silicates that ultimately return to the Earth’s surface with production of CO2 – which then returns to the atmosphere via volcanoes (Berner & Berner, 1996). The global rate of silicate weathering (plus the basalt seawater reaction) and of deposition of biogenic silica in marine sediments is in excess of 200 Tg Si (7 Tmol Si) per year.

Converting silicic acid to silica in higher plants While the predominant role of photosynthetic organisms in the silicon cycle is in converting silicates to silicic acid on land and converting silicic acid to silica in the ocean, terrestrial higher plants are also involved in the conversion of silicic acid to silica (Raven, 1983; Datnoff et al., 2001). Silicic acid enters the plants, as does water, and is carried in

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the transpiration stream toward transpiration termini (Canny, 1994). As water evaporates, silicic acid becomes supersaturated with respect to solid hydrated silica, which is precipitated as phytoliths. Ultimately these phytoliths are resolubilized and join the flux of silicic acid to the ocean. It is these silica deposits which give the increased resistance to grazers and parasites and changed leaf posture in many of the plants already mentioned (Datnoff et al., 2001). The quantity of silica that is deposited per unit dry matter gained depends on the quantity of silicic acid per unit water transpired and the quantity of water transpired per unit dry matter gain.

Movement through the plant Ma et al. (2001) point out that a ‘typical’ soil solution concentration of silicic acid of 0.35 mol m−3, and 500 g water transpired per g dry matter increase in a C3 plant, would yield 5 mg Si per g dry matter if the silicic acid and water were taken up in the same proportion as in the soil solution. Ma et al. (2001) measured the Si content of the dry matter of a range of terrestrial embryophytes, and compared the values with the 5 mg Si per dry matter that they had calculated for proportional uptake of silicic acid and water from the soil solution. Of the plants that they tested, Ma et al. (2001) found that bryophytes (two species), lycopsids (two species), sphenopsids (two species), pteropsids (26 species), eight species from the Cucurbitales, five species from the Urticales, one species from the Eriocaulales, seven species from the Cyperales and 211 species from the Graminales had Si contents of at least 10 mg Si per g dry matter. These plants are silicon accumulators or, in the case of the Cucurbitales and Urticales, in an intermediate category between nonaccumulaters and accumulaters as judged from the relatively low Si : Ca ratio in the plants (Ma et al., 2001). The plants tested by Ma et al. (2001), which were nonaccumulators of silicon, were 25 species of pteropsids, 12 species of gymnosperms, and two species from the Cyperales. The ‘nonaccumulators’ actually exclude silicic acid from the plant, because they contain less silicon than would be expected if there was nonselective passive entry of silicic acid with water. By contrast, the accumulators (and intermediate plants) take up silicic acid faster than would be expected from a nonselective entry of silicic acid with water during plant growth. The plants which exclude silicic acid all have endodermes in their roots (Raven & Edwards, 2001), and so could readily exclude silicic acid relative to water. This could occur regardless of whether these compounds enter by uncatalysed movement across the lipid component of the plasmalemma or, probably, when they enter by aquaporins (Raven, 2001; Tyerman et al., 2002). The plants which accumulate silicic acid must use active transport across (a) membrane(s). These plants do not always

have endodermes in their roots (e.g. the genus Lycopodium among Lycopsida) or other absorptive organs (e.g. belowground parts of bryophytes) (Raven, 2001; Raven & Edwards, 2001). This absence of an apoplasmic barrier would limit the extent to which leakage back to the medium of solutes actively transported into the transpiration stream could be prevented (Raven & Edwards, 2001; Raven, 2001). Notwithstanding this problem, active transport must occur in these cases. For higher plants, the active transport occurs inwards at the plasmalemma of root epidermal or cortical cells (or mycorrhizas?) and/or outwards at the plasmalemma abutting on xylem elements. While there is a good understanding at the molecular genetic level of silicic acid active influx in diatoms (Hildebrand et al., 1997; Hildebrand et al., 1998), much less is known about silicic acid active transport in higher plants.

Active transport of silicic acid in rice In this issue, Tamai & Ma report on results that significantly advance our understanding of the mechanism of active influx of silicic acid into roots of rice. Their work on net uptake by whole plants confirmed that rice takes up silicic acid (on a root dry matter basis) an order of magnitude faster than any of the other six cereal species tested. The lack of effect of pretreatment with silicic acid led the authors to conclude that the silicic acid transport system was constitutive rather than inducible. The transporter taking up silicic acid has a relatively low affinity (half-saturation of 0.32 mmol m−3). Studies with inhibitors suggest that aquaporins are probably not involved in silicic acid transport, and that anion antiporters are almost certainly not involved. Inhibitor studies also suggest that the catalytic site(s) of the transporter involves cysteine but not lysine residues. This work, together with that of Ma et al., 2003, on a rice mutant which is defective in silicic acid uptake, provides a good basis for further work.

Where now? Clearly we need more knowledge of how active transport of silicic acid occurs in higher plants. Especially important is better understanding of the high accumulation of silicon seen in rice, the single most important human food crop. In many rice-growing areas yields are significantly enhanced by fertilization with silicon fertilizers, in some cases as a result of prolonged silicon removal in the harvested crop (Datnoff et al., 2001). Finally, it is salutary to note that grasses and diatoms, with, respectively, a beneficial and an essential role for silicic acid, are very important in global net primary productivity. Grasses fix c. 15 Pg C per year out of c. 60 Pg C per year of net primary production on land, and diatoms fix > 15 Pg C per year out of c. 50 Pg C per year of net primary production in the

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ocean (Ajtay et al., 1979; Falkowski & Raven, 1997; Field et al., 1998). John A. Raven Division of Environmental and Applied Biology, School of Life Sciences University of Dundee, Dundee DD1 4HN, UK (tel +44 1382344281; fax +44 1382344275; email [email protected])

References Ajtay GL, Ketner P, Duvigneaud P. 1979. Terrestrial primary production and biomass. In: Bolin B, Degens T, Kempe S, Ketner P, eds. The global carbon cycle. Scope 13. Chichester, UK: John Wiley and Sons, 129–181. Berner EU, Berner RA. 1996. Global environment. Water, air and geochemical cycles. Uppersaddle River, NJ, USA: Prentice Hall. Britez RM, Watanabe T, Jansen S, Reissmann CB, Osaki M. 2002. The relationship between aluminium and silicon accumulation in leaves of Faramea merginata (Rubiaceae). New Phytologist 156: 437– 444. Canny MJ. 1994. What becomes of the transpiration stream? New Phytologist 114: 341–368. Datnoff LE, Snyder GH, Korndörfer GH, eds. 2001. Silicon in agriculture. Studies in plant science, 8. Amsterdam, The Netherlands: Elsevier. De Master DJ. 2002. The accumulation and cycling of biogenic silica in the Southern Ocean: revisiting the marine silica budget. Deep-Sea Research Plant II – Topical Studies in Oceanography 49: 3155–3167. Epstein E. 1999. Silicon. Annual Review of Plant Physiology and Plant Molecular Biology 50: 641–664. Falkowski PG, Raven JA. 1997. Aquatic photosynthesis. Malden, MA, USA: Blackwell Science. Field CB, Behrenfeld MJ, Randerson JT, Falkowski P. 1998. Primary production of the biosphere: Integrating terrestrial and oceanic compounds. Science 281: 237–246. Hamm CE, Merkel R, Springer O, Kurkoje P, Maler C, Prechtel K, Smetacek V. 2003. Architecture and material properties of diatom shells provide effective mechanical protection. Nature 421: 841– 843. Hildebrand M, Dahlin K, Volcani BE. 1998. Characterization of a silicon transporter family in Cylindrotheca fusiformis. Sequences, expression analysis, and identification of homologs in other diatoms. Molecular and General Genetics 260: 480–486. Hildebrand M, Volcani BE, Grossman W, Schroeder J. 1997. A gene family of silicon transporters. Nature 385: 688–689. Lucas Y. 2001. The role of plants in controlling rates and products of weathering: importance of biological pumping. Annual Review of Earth and Planetary Sciences 29: 135–163. Lux A, Luxova M, Abe J, Tanimoto E, Hattori T, Inanaga S. 2003. The dynamics of silicon deposition in the sorghum root endodermis. New Phytologist 158: 437–441. Ma JF, Miyake Y, Takahashi E. 2001. Silicon as a beneficial element for crop plants. In: Datnoff LE, Snyder GH, Korndörfer GH, eds. In: Silicon in agriculture. Studies in plant science, 8. Amsterdam, The Netherlands: Elsevier, 17–39.

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Ma JF, Tamai K, Ichii M, Wu G. 2003. A rice mutant defective in Si uptake. Plant Physiology 132: (In press.) Raven JA. 1983. The transport and function of silicon in plants. Biological Reviews 58: 179–207. Raven JA. 2001. Silicon transport at the cell and tissue level. In: Datnoff LE, Snyder GH, Korndörfer GH, eds. Silicon in agriculture. Studies in plant science, 8. Amsterdam, The Netherlands: Elsevier, 41–55. Raven JA, Edwards D. 2001. Roots: Evolutionary origins and biogeochemical significance. Journal of Experimental Botany 52: 381– 401. Tamai K, Ma JF. 2003. Characterization of silicon uptake by rice roots. New Phytologist 158: 431–436. Tyerman SD, Niemietz CM, Bramley H. 2002. Plant aquaporins: multifunctional water and solute channels with expanding roles. Plant, Cell & Environment 25: 173–194. Key words: silicon, active transport, diatoms, grasses, rice, silica, silicic acid, weathering. Commentary 158

Woody plants, carbon allocation and fine roots Sitting on the veranda, the sun reflects off the Mosel and the long rows of Vitis vinifera sloping down to the river. It’s a lazy autumn afternoon and my mind drifts from the nuances of the Embden-Meyerhof-Parnas pathway to the beautiful, neatly manicured rows of grape vines. The leaves are pale yellow and the physiological processes preceding the advent of the dormant season are unfolding. These vines are highly integrated physiological systems, with water, minerals, amino acids, carbohydrates, growth regulators, and other organic substances moving freely, though often in phases, between roots and shoots. How old are these vines? How deeply rooted are they, perched on this dry, southfacing slope? The ability of woody plants to survive for decades, centuries, sometimes millennia, is due in part to their capacity to withstand environmental stress by shifting their resources from roots, to shoots, to storage reserves. Grapes are no exception – vines can be in production for decades, and they survive the year-to-year vagaries of nature and horticultural manipulations designed to encourage higher berry yields by shifting resource allocation. In this issue of New Phytologist (pp. 489–501), Anderson et al. report how irrigation, pruning and annual variations in weather influence the survivorship of roots of Concord grape, Vitus labruscana. There are two interesting perspectives raised: • The importance of whole plant source–sink relationships in driving fine root lifespan. • Fine roots as modular plant organs with different life expectancies depending on various environmental and developmental factors.

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Carbon allocation to roots Interest in the form and function of woody plant root systems has grown tremendously in recent years. The structural (‘woody’) portion of the root system serves important transport and storage functions and these roots can penetrate several meters vertically into the soil (Nepstad et al., 1994). Experimental removal of woody shrubs from semiarid regions of the world results in an increase in water yield from entire watersheds, and carbon allocation to roots plays a significant role in the global carbon cycle (Jackson et al., 1997, 2000). Some of the most widely applied forest productivity models are now calibrated by allocating carbohydrates to roots first in recognition of the fact that root to shoot relationships fundamentally control productivity at the species level (Landsberg et al., 2003). Whole plant source–sink relationships exhibit strong seasonal rhythms and respond to defoliation, pruning and stress. Prominent among the functions ascribed to roots is their role in the storage of carbohydrate reserves. In fact, the root systems of woody plants typically contain higher concentrations of reserve carbohydrates than the stem system (Loescher et al., 1990).

The seasonal cycle of carbohydrate reserves There are numerous reports of seasonal variations of carbohydrate reserves in roots, which provide indirect evidence for the role of these substances in woody plant growth. The general pattern is for root reserves to decline, often quite rapidly, just before or with the onset of the growing season, when shoots and roots are rapidly expanding. Then, when shoots are fully refoliated, reserves begin to build back up to preflush levels, reaching a maximum early in the dormant season. Although variation occurs among species, this general pattern has been consistent among diverse taxa (Dickmann & Pregitzer, 1992). Anderson et al. (2003) demonstrate that roots produced before bloom in the spring have the shortest lifespan and they speculate that this may be caused by lower carbohydrate reserves. The strongest direct evidence for the seasonal cycling of root carbohydrate reserves comes from 14C studies. If leaves are exposed to 14CO2 late in the growing season, the tracer is transported to the root system, as well as to sites of branch and stem storage (Kandiah, 1979; Lippu, 1998). The key regulatory event that shifts translocation of carbohydrate on a particular shoot axis basipetally to the root system appears to be bud set (Isebrands & Nelson, 1983). During rapid shoot elongation little carbohydrate build-up occurs in the roots. As a particular shoot axis ceases growth and sets buds, particularly if it is removed from still active vegetative and reproductive sinks (Quinlan, 1969), basipetal translocation to the lower stem and roots predominates. A great deal of

the 14CO2 assimilated in autumn is stored as reserves in the root system (Lippu, 1998). Nguyen et al. (1990) reported that starch concentrations in the fine roots of one Populus genotype increased an astonishing 75 times between September and November. Thus, in autumn, there is a strong downward pulse of nonstructural carbohydrates, which are stored in the root system during the dormant season. Interestingly, most of these reserves appear to be used primarily for root maintenance respiration during the dormant season and new shoot growth the following spring (Lippu, 1998). Fine root growth in the spring appears to be fuelled primarily by current photosynthate, not dormant season carbohydrate reserves (van den Driessche, 1987; Lippu, 1998). However, the role of seasonal source–sink relationships and the utilization of stored reserves in determining rates of root growth and lifespan are not well understood and we await more definitive studies from mature plants. Much of what we know comes from young fruit trees and nursery-sized conifers. It has never been easy to use 14CO2 in the field on mature woody plants. Thankfully, 13CO2 as a tracer has a bright future (Ehleringer et al., 2000), and it should be very useful in field situations where 14CO2 is always problematic. The implementation of 13CO2 experiments explicitly designed to understand transient physiological processes – such as the seasonal storage and remobilization of carbohydrate reserves in woody plants – should prove fruitful in the future.

Shoot sink strength alters root mortality Carbon allocation patterns are very complex in perennial plants because of the multiple-age structure of leaves and roots. Farrar & Jones (2000) examined four hypotheses related to carbohydrate allocation and concluded that the ‘shared-control’ hypothesis was most consistent with empirical data from a number of studies. Anderson et al. (2003) report that higher grape yields and pruning increase the risk of fine root mortality. In both cases, there is obvious indirect evidence that the sink strength of the shoot system can directly influence the lifespan of fine roots. Exposure of plants to ozone directly reduces photosynthetic capacity and shifts carbohydrate allocation to the repair of the shoot system. This in turn results in a decrease in carbon allocation to roots and mycorrhizas and increases fine root mortality (Anderson, 2003). In many cases decreased allocation to roots in response to ozone exposure occurs quickly (Gorissen & van Veen, 1988). It seems safe to conclude that the lifespan of fine roots can be very dynamic and depends fundamentally on whole plant patterns of carbon allocation. Increasing shoot sink strength can result in decreased fine root lifespan. The challenge seems rather obvious – we need to understand the whole plant. Isolated studies of shoot and root systems will not be as profitable as integrated studies of whole plants.

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Fine roots as plant modules The fine roots of mature woody plants are ephemeral plant modules (sensu Harper, 1977) which arise from adventitious buds. Populations of fine roots can be studied using classical demographic techniques and they have a life cycle with different probabilities of transition from one physiological state to another (Hendrick & Pregitzer, 1992). In recent years we have learned that specific root length, nitrogen concentration, and rate of root respiration increase from the proximal to the distal end of the fine root system (Pregitzer et al., 1997, 2002; Burton et al., 2002). The root tips are metabolic ‘hot spots’ and of course this is also the primary point of association with mycorrhizas. Wells & Eissenstat (2001), Wells et al. (2002), and now Anderson et al. (2003) have shown that small diameter roots have a higher risk of mortality (shorter lifespan) than larger diameter roots. In other words, the more you migrate toward the distal end of the lateral fine root system, the more active the root is physiologically and the greater the risk of mortality (the shorter the average lifespan). Pregitzer et al. (2002) report putative lateral fine root ‘branch scars’ along the perennial roots of numerous North American trees. The obvious hypothesis is that fine roots have preprogrammed points of ‘abscission’, but this idea remains untested. The focus on roots themselves is, however, too ‘phyto-centric’. Mycorrhizas are strong sinks for plant carbohydrates and King et al. (2002) report that mycorrhizal roots have a significantly lower risk of mortality than nonmycorrhizal roots (Pregitzer, 2002). Lifespans of metabolically active fine roots at the distal end of the branching root system must be all about carbohydrate sink strength (Anderson, 2003).

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what in the world is the ‘modular unit’ under consideration? Since we often observe only a part of the fine root system, for example in minirhizotrons, we have yet to develop a complete understanding of how fine roots are assembled and how they die. It would be useful if plant anatomists took an interest in this fundamental issue. Second, it is possible that different roots on the same root system have different primary functions, for example, water vs nitrogen uptake (Gebauer & Ehleringer, 2000). If this phenomenon were common, fine root structure, lifespan and physiology might well depend on the essential soil resource being acquired. After all, plants have more than one problem to solve in the soil (e.g. water, nitrogen, phosphorus). Finally, mycorrhizas are yet another interesting wildcard. Do some roots team up with certain mycorrhizas to acquire a specific essential soil resource, altering their branch structure, lifespan and physiology in the process? We still don’t understand the structure, lifespan and physiology of lateral fine root branches, but Anderson et al. have raised many, very interesting questions.

Acknowledgements This work was supported by the Division of Environmental Biology (Ecosystem Studies) of the National Science Foundation, the Office of Biological and Environmental Research (BER) and NIGEC of the Department of Energy, and the USDA Forest Service Northern Global Change Research Program and the North Central Research Station. Without this support, our research would not be possible and I am grateful for continued support. Kurt S. Pregitzer

The take home message What is the take home message? It seems likely that lateral fine root branches exhibit as much variability in form and function as we see in shoot systems (Reich et al., 1999; Wright & Westoby, 2002). Perhaps fine root form and function are directly related to leaf structure, lifespan and physiology – as Grubb (2002) points out, we need to turn our attention to coordinated studies of leaf and root properties. However, when it comes to fine roots, there are serious points of confusion. First, we do not know how to describe the basic plant module. Should we focus on diameter, position of a root segment on the branching root system, or try to understand how lateral fine root branches are constructed? Clearly, it will be difficult to compare the structure, lifespan and physiology of fine roots among plant taxa if we can’t decide how to describe the basic sampling unit. Anderson et al. (2003) seem to advocate a focus on diameter, but I believe we need to understand the structure, lifespan and physiology of entire lateral fine root branches from the point where they arise from adventitious buds. Just

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School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA (tel +1 906 4872396; fax +1 906 4872915; email [email protected])

References Anderson CP. 2003. Source-sink balance and carbon allocation below ground in plants exposed to ozone. New Phytologist 157: 213 – 228. Anderson LJ, Comas LH, Lakso AN, Eissenstat DM. 2003. Multiple risk factors in root survivorship: a four-year study in Concord grape. New Phytologist 158: 489 –501. Burton AJ, Pregitzer KS, Ruess RW, Hendrick RL, Allen MF. 2002. Root respiration in North American forests: effects of nitrogen concentration and temperature across biomes. Oecologia 131: 559 –568. Dickmann DI, Pregitzer KS. 1992. The structure and dynamics of woody plant root systems. In: Mitchell CP, Ford-Robertson JB, Hinkley T, Sennery-Forsse L, eds. Ecophysiology of short rotation forest crops. New York, USA: Elsevier Applied Science, 95 –123.

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Ehleringer JR, Buchmann N, Flanagan LB. 2000. Carbon isotope ratios in belowground carbon cycle processes. Ecological Applications 10: 412– 422. Farrar JF, Jones DL. 2000. The control of carbon acquisition by roots. New Phytologist 147: 43 –53. Gebauer RLE, Ehleringer JR. 2000. Water and nitrogen uptake patterns following moisture pulses in a cold desert community. Ecology 81: 1415 –1424. Gorissen A, van Veen JA. 1988. Temporary disturbance of translocation of assimilates in Douglas firs caused by low levels of ozone and sulfur dioxide. Plant Physiology 88: 559 –563. Grubb PJ. 2002. Leaf form and function – towards a radical new approach. New Phytologist 155: 317– 320. Harper JL. 1977. Population biology of plants. London, UK: Academic Press. Hendrick RL, Pregitzer KS. 1992. The demography of fine roots in a northern hardwood forest. Ecology 73: 1094 –1104. Isebrands JG, Nelson ND. 1983. Distribution of 14C-labelled photosynthates within intensively cultured Populus clones during the establishment year. Physiological Plant 59: 9 –18. Jackson RB, Schenk HJ, Jobbagy EG, Canadell J, Colello GD, Field CB, Dickinson RE, Friedlingstein P, Heimann M, Kleidon A, Hibbard K, Kicklighter DW, Neilson RP, Parton WJ, Sala OE, Sykes MT. 2000. Belowground consequences of vegetation change and their treatment in models. Ecological Applications 10: 470 – 483. Jackson RB, Mooney HA, Schulze E-D. 1997. A global budget for fine root biomass, surface area, and nutrient contents. Proceedings of the National Academy of Sciences, USA 94: 7362 –7366. Kandiah S. 1979. Turnover of carbohydrates in relation to growth in apple trees. II. Distribution of 14C assimilates labeled in autumn, spring and summer. Annals of Botany 44: 185 –195. King JS, Albaugh TJ, Allen HL, Buford M, Strain BR, Dougherty P. 2002. Below-ground carbon input to soil is controlled by nutrient availability and fine root dynamics in loblolly pine. New Phytologist 154: 389 –398. Landsberg JJ, Waring RH, Coops NC. 2003. Performance of the forest productivity model 3-PG applied to a wide range of forest types. Forest Ecology and Management 172: 199 –214. Lippu J. 1998. Redistribution of 14C-labelled reserve carbon in Pinus sylvestris seedlings during shoot elongation. Silva Fennica 32: 3 –10.

Loescher WH, McCarmant T, Keller JD. 1990. Carbohydrate reserves, translocation, and storage of woody plant roots. Horticultural Science 25: 274 –281. Nepstad DC, de Carvalho DR, Davidson EA, Jipp PH, Lefebvre PA, Negreiros GH, da Silva ED, Stone TA, Trumbore SE, Vieira S. 1994. The role of deep roots in the hydrological and carbon cycles of Amazonian forests and pastures. Nature 372: 666 – 669. Nguyen PV, Dickmann DI, Pregitzer KS, Henrick RL. 1990. Late-season changes in allocation of starch and sugar to shoots, coarse roots and fine roots in two hybrid poplar clones. Tree Physiology 7: 95 –105. Pregitzer KS. 2002. Fine roots of trees – a new perspective. New Phytologist 154: 267–270. Pregitzer KS, Deforest JL, Burton AJ, Allen MF, Ruess RW, Hendrick RL. 2002. Fine root architecture of nine North American trees. Ecological Monographs 72: 293 –309. Pregitzer KS, King JS, Burton AJ, Brown SE. 2000. Responses of tree fine roots to temperature. New Phytologist 147: 105 –115. Pregitzer KS, Kubiske ME, Yu CK, Hendrick RL. 1997. Relationships among root branch order, carbon, and nitrogen in four temperate species. Oecologia 111: 302 –308. Quinlan JD. 1969. Mobilzation of 14V in the spring following autumn assimilation of 14CO2 by an apple rootstock. Journal of Horticultural Science 44: 107 –110. Reich PB, Ellsworth DS, Walters MB, Vose JM, Gresham C, Volin JC, Bowmann WD. 1999. Generality of leaf trait relationships: a test across six biomes. Ecology 80: 1955 –1969. Van den Driessche R. 1987. Importance of current photosynthate to new root growth in planted conifer seedlings. Canadian Journal of Forest Research 17: 776 –782. Wells CE, Eissenstat DM. 2001. Marked differences in survivorship among apple roots of different diameters. Ecology 82: 882 – 892. Wells CE, Glenn DM, Eissenstat DM. 2002. Changes in the risk of fine root mortality with age: a case study in peach, Prunus perscia (Rosaceae). American Journal of Botany 89: 79 – 87. Wright IJ, Westoby M. 2002. Leaves at low versus high rainfall: coordination of structure, lifespan and physiology. New Phytologist 155: 403 – 416. Key words: Concord grape (Vitus labruscana), fine roots, woody plant physiology, root lifespan, carbon allocation.

Meetings Healthy soils, healthy people

International workshop on plant–soil interactions, Beijing, P.R. China, October 2002.

Rhizosphere, preferential flow and bioavailability: a holistic view of soil-to-plant transfer, Ascona, Switzerland, September 2002.

Increasing worldwide recognition of the need for ‘healthy soil’ with respect to plant biodiversity and productivity, and hence human wellbeing, is spawning a range of important conferences. Multidisciplinary and – not the same thing

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– interdisciplinary aspects of the soil–plant continuum need discussion and two recent conferences dealing with important aspects of rhizosphere science and plant growth focussed on this imperative. The location of the second of these meetings, in China, is significant. While attracting an international audience, the meeting also had many delegates from within the country and this regional perspective was a vital element – the sheer size of China and the diversity of its soils, climate and vegetation meant that new research areas were brought before the international community. Science in China is still often neglected by the rest of the world, and yet the potential, contrasting its environmental problems against its economic goals, is massive. The ‘problem soils’, and urgent need for revegetation and production of high-quality food for animals and humans presents an exciting scientific challenge.

Metals and rhizosphere toxicity The primary interests at the Ascona workshop lay within chemical and physical aspects of soil science, rhizosphere microbiology (bacteria and mycorrhizal fungi) and plant science, especially root research (details are available at http:// www.ito.umnw.ethz.ch/workshop.monteverita/program.htm). The keynote addresses, together, gave an integrated view of transfer processes and highlighted research needs at the interfaces between the biology, chemistry and physics of soils. Stephan Krämer (ETH Zürich, Switzerland) started by discussing rhizosphere biogeochemical processes and the availability of trace metals to plants, a theme taken up later on, by Thomas Kuyper (Wageningen University, The Netherlands), who proposed that Al-tolerant maize only acquires P from acidic tropical soils through mycorrhizal associations – something that plant biotechnologists might well note. Other highlights included the presentation by Jan Jansa and coworkers (ETH Eschikon, Switzerland), of phosphorus and zinc flows through hyphae of arbuscular mycorrhizal fungi growing in root-free compartments; and the account by Tiziana Centofanti (ETH Zürich, Switzerland) of her preliminary results in a study of 65Zn, 54Mn, 57Co and 134Cs uptake by maize under field and pot conditions, as related to heterogeneous distribution of the radiotracers and plant roots. Indeed, delegates from at the meeting in China covered an enormous range of rhizosphere toxicities, including selenium (Weixuan Fang, Institute of Geochemistry, Chinese Academy of Sciences, and the Mineral & Geological Exploration Center, Beijing), arsenic (e.g. Mei Lei & Xiaoyong Liao, Institute of Geographic Sciences and Natural Resources Research, CAS, Beijing; see also Meharg & Hartley-Whitaker, 2002; Meharg, 2002), cadmium (e.g. Kairong Wang, Huazhong Agricultural University, Wuhan, and the Institute of Subtropical Agriculture, CAS, Changsha; see also McGrath et al., 2001), copper (e.g. Jing Song,

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Institute of Soil Science, CAS, Nanjing), and several others. Peter Christie (Queen’s University, Belfast, UK) assessed the potential for mycorrhizas to alleviate heavy metal toxicity – an area tackled in collaboration with Xiaolin Li and his group at the China Agricultural University in Beijing. Another notable contribution was by Mitsuru Osaki (Hokkaido University, Sapporo, Japan), who described strategies to improve rhizosphere processes in the saline and acidic soils of Kalimantan, Indonesia, which have been disturbed by agricultural development activities.

Roots and mycorrhizas Mycorrhizas and root nutrient uptake, naturally, formed a common thread running through both meetings. In addition to the heavy metal work already mentioned by several researchers, Marcel Bucher described some of the exciting work on mycorrhiza-specific phosphate transporters being done by his group at Eschikon (Switzerland). In Beijing, a session dealt with adaptations that influence phosphate uptake by plants growing in low-phosphate soils, and Sally Smith (University of Adelaide, Australia) described functional diversity between arbuscular mycorrhizal fungi revealed in studies of the external mycelium. Mike Miller (Agronne National Laboratory, USA) ably showed, in one of the meeting’s highlights, how arbuscular mycorrhizal fungi can influence soil structure. Hans Lambers (University of Western Australia), emphasized the role of cluster roots of constitutively nonmycorrhizal plants as an adaptation to extremely phosphate impoverished soils and Xialong Yang (South China Agricultural University, Guangzhou) discussed genetic variation in root-hair traits regulated by plant phosphorus status.

Modelling At Ascona, Sylvain Pellerin (INRA, Unité d’Agronomie, Villenave d’Ornon, France) told us of lessons learned from modelling plant uptake of P and other nutrients, and the modelling theme resurfaced later on – the last keynote address by Brent Clothier (HortResearch, Palmerston North, New Zealand) returned superbly to the holistic ‘transfer’ theme in ‘Measuring and modelling the leakiness of the root-zone to contaminants; towards a unified model’. The workshop also included discussions focussed on seeking ways of integrating individual knowledge with modelling tools – the utility of such models for nutrient transfer was controversial.

Soil What of the soil itself? At Ascona, Alain Pierret (CSIRO Land & Water, Canberra, Australia) spoke on recent developments in quantification of three-dimensional

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structures, investigating the interactions of roots and soil structure – these developments are progressively increasing the resolution of the structures that can be observed. Soil water relations were also well covered, especially with respect to heterogeneity in soil structure as discussed by Andreas Greiffenhagen (Technical University of Berlin, Germany): ‘Studies on soil water fluxes in pine forests’. Returning to human well-being, agriculture is of paramount importance, and at the Beijing workshop Fusuo Zhang (China Agricultural University, Beijing) described beneficial rhizosphere processes, covering rhizosphere management towards sustainable agriculture.

Conclusions The complexities of rhizosphere science are such that conferences such as these, and the three New Phytologist Symposia held earlier in 2002 (e.g. Smith, 2002), are increasingly important in complementing the very large international conferences that focus on organisms (e.g. plants or fungi), symbioses generally (or mycorrhizas in particular), processes (e.g. the large field of plant nutrition), and soil science. They allow good contact between delegates, irrespective of ‘status’. The opportunity to emphasize national or regional research priorities, often lost at very large international conferences, is attractive to researchers and sponsors alike. Swiss-based initiatives, in introducing more realistic multicomponent models for soil–plant transfer processes, will have benefits for research done in other plant ecosystems, both natural and managed. The major soil problems in China, some quite new but others a result of centuries and even millennia of mining activities, will doubtless receive increasing attention from researchers worldwide. If these conferences are anything to go by, international networks of rhizosphere scientists will certainly flourish.

Acknowledgements The Ascona meeting was held in the Centro Stefano Franscini

on the ‘Mountain of Truth’: Monte Verità, and was sponsored by the Swiss Federal Institute of Technology (Eidgenössische Technische Hochschule: ETH), Zürich. The organisers, headed by Stephan Krämer, the technical staff, and the staff at the Centro Stefano Franscini, did a superb job. The International Workshop on Plant–Soil Interactions was held under the auspices of the Research Centre for EcoEnvironmental Sciences (RCEES) of the Chinese Academy of Sciences (CAS) and the University of Adelaide, Australia. Nearly all the organisation was done in Beijing by a group led by the energetic Yongguan Zhu. The Chinese Academy of Sciences, RCEES and its Director General, Jingzhu Zhao, Yongguan Zhu, and (not least) his team of helpers, well deserved the thanks of all who attended. During the workshop the joint RCEES–University of Adelaide laboratory – a brain-child of Prof. Zhu – was officially opened. I thank Jonathan Ingram for editorial help in integrating the themes of these meetings. F. Andrew Smith Soil and Land Systems, School of Earth and Environmental Sciences, The University of Adelaide, SA 5005, Australia (tel +61 883036517; fax +61 883036511; email [email protected])

References McGrath SP, Lombi E, Zhao F-J. 2001. What’s new about cadmium hyperaccumulation? New Phytologist 149: 2–3. Meharg AA. 2002. Arsenic and old plants. New Phytologist 156: 1–8. Meharg AA, Hartley-Whitaker J. 2002. Arsenic uptake and metabolism in arsenic reistant and nonresistent plant species. New Phytologist 154: 29–43. Smith SE. 2002. Soil microbes and plants – raising interest, mutual gains. New Phytologist 156: 142–144. Key words: soil, mycorrhizas, rhizosphere, bioavailability, heavy metals, plant–soil interactions. Letters 158

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Letters Stomatal precursors in Arabidopsis: prohibiting the fulfilment of a general rule More than 100 years ago, biologists realised that knowing the shape of a cell or its direction of expansion, the orientation of its future cell division plane can be predicted. Hofmeister and Errera formulated general rules for cell division orientation. Here, we report on an investigation into the fulfilment of these general rules during stomatal formation in the hypocotyl of Arabidopsis. Unexpectedly, we found the first exception for a symmetric division to the Hofmeister’s and Errera’s rules

Hofmeister’s and Errera’s rules In plants, where cell migration cannot take place, the orientation of the cell division planes plays a crucial role in defining the future pattern of cells and structures. It is known that the shape of a cell or its direction of expansion allows a prediction to be made of the precise orientation of its division plane and therefore the position of the daughter cells in the plant body (Smith, 2001). In 1863, Hofmeister formulated a rule that cells divide in a plane perpendicular to the expansion axis (Hofmeister, 1863; Fig. 1(a). Errera pointed out 25 yr later that cell division takes place so that the cell division plane has the minimal area for producing two daughter cells of similar sizes (Errera, 1888; Fig. 1(a). Direct evidence supporting these rules comes from elegant cell deformation experiments in which cells divided, following Hofmeister’s and Errera’s rules, in a plane parallel to the compressive force (Lintilhac & Vesecky, 1984; Lynch & Lintilhac, 1997). Asymmetric cell divisions are remarkable exceptions to these general rules (Smith, 2001). In the Arabidopsis hypocotyl (embryonic stem), epidermal cells are organised into files that run parallel to the long axis of the seedling, providing a highly ordered division plane network. Stomatal development progresses from the apical to the basal pole of this embryonic organ (Berger et al., 1998), starting when hypocotyl elongation approaches completion (Gendreau et al., 1997). The number and orientation of cell divisions that the stomatal precursors undergo

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Fig. 1 The guard mother cell (GMC) division plane does not follow the Hofmeister’s and Errera’s rules. (a) A hypothetical oval cell dividing in a plane perpendicular to its long axis, as the Hofmeister’s and Errera’s rules predict. (b) The GMC division plane is not perpendicular to its long axis; > 90% of GMCs divide in a plane parallel to their main growth axis.

have been precisely described by Berger et al. (1998). The first sign that indicates the stomatal pathway initiation is a cell division along the longitudinal axis. One of the daughter cells undergoes one transverse cell division. In many stomatal complexes, this transverse cell division produces the guard mother cell (GMC), which divides symmetrically producing the stoma.

Microscopical analysis Seedlings of Arabidopsis thaliana (Landsberg erecta ecotype) were used. Seeds were vernalized at 4°C for several days and surface-sterilised in 5% sodium hypochlorite. They were plated on Petri dishes containing Murashige and Skoog salts (Sigma, St. Louis, MO, USA) supplemented with 1% sucrose and solidified with 1% agar. Seedlings were germinated and grown in vertically oriented dishes, at 22°C and in a 16- h-light/8- h-dark cycle. Light was provided from 12 cool-white fluorescent bulbs (TLD 30 W/ 33). Impressions of the epidermal surface in 17 hypocotyls were taken with 6% agarose as described (Mathur & Koncz, 1997). The first imprints were taken from 6 d old seedlings, which have a high number of GMCs. The second imprints were taken 4–5 d later. Imprints were mounted on slides, and examined and digitised under Nomarsky optics with a Leica DC 300F camera attached to a Leica DMIRB inverted

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Fig. 2 Serial imprints showing the orientation of two guard mother cell (GMC) division planes. (a) and (c) First imprints. (b) and (d) Second imprints of epidermal tissue represented in (a) and (c), respectively. In (a) and (b) the GMC divides equally and symmetrically, producing the two guard cells that compose the stoma. Note that the GMC division plane overlies its long axis. In (c) and (d), the GMC divides equally and symmetrically giving rise to two guard cells (GCs), but following its short axis. Note that, nevertheless, stomata formation takes place. As in (a), > 90% of GMCs divided. The red colour indicates cells whose fate is discussed. The yellow line indicates the long axis of GMCs (in (a) and (c)) and the orientation of the GMCs’ division plane (in (b) and (d)). Bar, 20 µm. All images are at the same magnification.

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microscope (Wetzlar). Images were processed using Photoshop 6.0 (Adobe Systems, Mountain View, CA, USA).

Breaking the rules In order to confirm the Hofmeister’s and Errera’s rules, we analysed the orientation of the GMC division plane in the Arabidopsis hypocotyl. Unexpectedly, of a total of 40 ovalshaped GMCs scored, 37 (91.9%) divided equally (in cell size) and symmetrically (in cell fate), but in a plane parallel (0° ± 15) to their long axis (Fig. 2a,b). The cell division plane is also parallel to the long axis of the organ. This reveals that ‘unknown factors’ not only prohibit the fulfilment of Hofmeister’s and Errera’s rules, but also impose a new rule for the alignment of the GMC division plane. The three (8.1%) remaining GMC division planes did not show any preferential orientation; they were oriented at 90° ± 15, 70° ± 15 and 45° ± 15, relative to the long axis of the hypocotyl. Nevertheless, all GMCs gave rise to stomata (Fig. 2c,d). This result indicates that a specific orientation of the GMC division plane is not required for stomata formation. Every GMC division produced two daughter cells of similar sizes, suggesting that although the orientation of the GMC division plane plays no role in stomatal fate determination and differentiation, the size of GMC daughter cells might be an essential requirement for this process. In other nonelongated organs, such as leaves and cotyledons of Columbia background, the GMC preprophase band circumscribes the long axis of the cell (Zhao & Sack, 1999). Considering that the preprophase band position directs the future localization of the cell division plane and the maturation of new cell walls (Mineyuki & Gunning, 1990), then the new rule imposed is not restricted to the hypocotyl GMCs of Landsberg erecta, but rather it seems to be a general feature of GMCs all over the plant organs and ecotypes. The selection of the proper cleavage plane orientation determines the relative positions of the daughter cells relative to their neighbouring cells. The finding that stomata formation does not depend on the GMC cleavage plane orientation suggests that signals from specific epidermal or mesophyll cells surrounding the stoma are not required for stomatal cell fate determination and differentiation. Intrinsic factors and/or long-distance signalling, as demonstrated for the latter for stomata formation in the Arabidopsis leaves (Lake et al., 2001), should then guide stomata formation in the embryonic stem. To our knowledge, GMC division is the first exception found for a symmetric division to the Hofmeister’s and Errera’s rules. Some asymmetric cell divisions also elude these rules. For example, the two subsidiary cells in maize leaves (Gallagher & Smith, 2000) and the first subsidiary cell in Arabidopsis hypocotyl (Berger et al., 1998) arise from cell divisions that are parallel to the main cell growth axis. This orientation might be crucial to produce an unequal

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segregation of cell fate determinants. In the GMC division, it could be argued that the new plane is also set to ensure the correct segregation of intrinsic factors; however, in the few cases in which the GMC division plane deviated from the expected position, the guard cells still formed correctly, which might rule out such a possibility. The unexpected orientation of the GMC division plane in the Arabidopsis hypocotyl is not only an exception to a general rule, but it also provides a model system to address the genetic and molecular control of the mechanism that regulates the orientation of the cell division plane during plant development.

Acknowledgements This work was supported by a grant from the Spanish National Funding Agency (DGESIC, project PB97-0024) to CF and by a research aid from the UCLM to LS and CF. We thanks Frederick D. Hempel for helpful discussions. Laura Serna* and Carmen Fenoll Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain *Author for correspondence (tel +35 925 26 88 00, ext. 5467; fax +34 925268840; email [email protected])

References Berger F, Linstead P, Dolan L, Haseloff J. 1998. Stomata patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Developmental Biology 194: 226 –234. Errera L. 1888. Uber Zellfromen und Seifenblasen. Botanisches Centralblatt 34: 395–398. Gallagher K, Smith LG. 2000. Roles for polarity and nuclear determinants in specifying daughter cell fates after an asymmetric division in the maize leaf. Current Biology 10: 1229–1232. Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M, Höfte H. 1997. Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiology 114: 295–305. Hofmeister W. 1863. Zusütze und Berichtigungen zu den 1851 verüffentlichen Untersuchungen der Entwicklung hüherer Kryptogamen. Jahrbücher für Wissenschaftliche Botanik 3: 259–293. Lake JA, Quick WP, Beerling DJ, Woodward I. 2001. Signals from mature to new leaves. Nature 411: 154. Lintilhac PM, Vesecky TB. 1984. Stress-induced alignment of division plane in plant tissues grown in vitro. Nature 307: 363–364. Lynch TM, Lintilhac PM. 1997. Mechanical signals in plant development: a new method for single cell studies. Developmental Biology 181: 246–256. Mathur J, Koncz C. 1997. Method for preparation of epidermal imprints using agarose. Biotechniques 22: 280–282. Mineyuki Y, Gunning BES. 1990. A role for preprophase bands of

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microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. Journal of Cell Science 97: 527–537. Smith LG. 2001. Plant cell division: Building walls in the right places. Nature Reviews Molecular Cell Biology 2: 33–39. Zhao L, Sack F. 1999. Ultrastructure of stomatal development in

Arabidopsis (Brassicaceae) leaves. American Journal of Botany 86: 929–939. Key words: Arabidopsis thaliana, hypocotyl, stomata, guard mother cell, cell division, serial imprints, Hofmeister’s rule, Errera’s rule.

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