how do real roots - plant physiology · roots on the corn plants shown in figures 1a and 2, only...

Plant Physiol. (1995) 109: 1-6

How Do Real

Some New Views

Roots Work?’

of Root Structure

Margaret McCully*

Department of Biology, Carleton University, Ottawa, Canada K1 S 5B6

Root systems of plants growing in the field are marvel- ously successful at foraging for nutrients and water in a hostile, competitive environment where supplies of them are very limited, local, and variable. This success is not surprising because this is the environment in which they have evolved. We are uncovering increasing complexities of this life of roots in the soil, such as their interactions with a broad range of microbes well beyond the better-known relationships with mycorrhizal fungi and root-nodule bacteria. More is being discovered about the two-way inter- change of materials and messages across the root-soil interface. Growth movements, the groping for nutrients and water, have intrigued plant biologists since they were so well described by Darwin, and they continue to be puzzles that become more complex as they are better understood. Additionally, the variety and success of responses of roots to environmental stresses multiply and raise more puzzling questions. Useful background material concerning these topics is available in Waisel et al. (1991).

Despite these advances, the model of root structure used to interpret root system function is inadequate. This model is too often the stylized, simplistic, textbook interpretation of old studies of seedling roots of a very few species and is derived from roots and shoots still living heterotrophically on reserves, transpiring little or not at all. Also, these roots have never met the rigors of the field soil. Furthermore, traditional anatomical study has focused on the apical meristem and the patterns of the young tissues within a few millimeters of the tip, rarely beyond a few centimeters. One laboratory manual, a standard reference for almost 30 years, includes a table of the distances from the root tip at which major developmental events occur in seven different species. The farthest distance indicated is 8.3 cm in a woody plant. For the herbaceous species, the last developmental event is even closer to the tip. Corn xylem lignification is listed at less than 1.7 cm. The limitations of such a view can be calculated from Dittmer’s (1937) famous data for total lengths of roots (main and three orders of branches) of a single soil-grown rye plant. The first 10 cm of a11 143 main roots total about one millionth of the whole root length.

’ This work was supported by an operating grant from the

* E-mail [email protected]; fax 1-613-788-4497. Natural Sciences and Engineering Research Council of Canada.

In 1980, together with a brave graduate student named Janet Vermeer, I began to study the roots of corn plants in the field. We started by washing the roots to get rid of as much of the “dirt” as possible, so worrying to an electron microscopist. But a freshly excavated plant, unwashed as shown in Figure 1 A, impressed us with the heterogeneity in size, origin, and soil binding, which have intrigued me and my students ever since. This interest has led us to explore the structure and functioning of root systems growing in the field. The results have been replete with surprises. In this paper I will include some of these surprises and the work of other groups, which provide new insights into three aspects of root structure and function, with important implications for many root-related studies: (a) delayed development of xylem, (b) the development of the interface with the soil, and (c) branch roots and their role.

DELAYED DEVELOPMENT OF XYLEM

Two types of primary xylem are involved in water and solute transport from roots to shoots in maturing plants: EMX and LMX. Early and late refer to the relative times (and positions along the roots) of the maturation of these tissues (i.e. of the death and union of the cells that make hollow tubes). Protoxylem, composed of very narrow, ephemeral tubes close to the root tips, probably does not deliver water and solutes to maturing shoots. In the older portions of the roots of many dicotyledons, a third type, secondary xylem (formed from a cambium), develops conducting tubes to deliver water and solutes. The LMX conducting tubes are always much wider than those of the EMX (Fig. 1B). This difference has a profound effect on their relative capacities to deliver solution to the shoot, because the volume of fluid delivered by a pipe varies as the 4th power of the radius. Thus, each of the large LMX tubes shown in the corn root in Figure 1B would carry about 5000 times the volume carried by each EMX tube. (Because there are about twice as many EMX tubes, the LMX system will actually deliver about 2500 times more solution than the EMX system.) In the soybean root in Figure lD, a similar calculation gives a ratio of about 100. Despite this dominance in volume flow, there has been

Abbreviations: EMX, early metaxylem; LMX, late metaxylem. 1 www.plantphysiol.orgon September 12, 2020 - Published by Downloaded from

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McCully Plant Physiol. Vol. 109, 1995

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Some New Views of Root Structure 3

neglect of LMX elements because they mature beyond the region where textbook descriptions cease. In dicotyledons the central parenchyma in this region of main roots shows no sign that it will subsequently develop into enormous LMX tubes. Paradoxically, in the monocotyledons the immature elements that will eventually form the LMX tubes are the first cells to enlarge in the stele behind the meristem and, therefore, have been believed to mature very early.

The simplest way to determine whether the LMX conducting tubes are open in any root region is to cut a length of the root and try to blow air through it into water. With the cortical tissue at the mouthpiece end removed, the only bubbles that you will be able to blow will be through the LMX tubes, because there is too much resistance in the much smaller EMX tubes. It was a variation of this proce- dure that first focused our attention on the LMX. Of the roots on the corn plants shown in Figures 1A and 2, only the fine, soil-free seminal roots yielded xylem sap to a hand-vacuum pump. The large, soil-covered roots, even with 10 cm of their tip removed, yielded no xylem sap and would hold the vacuum. This test on field-grown corn plants revealed that nowhere along the first 20 to 30 cm of a soil-covered root were the LMX tubes open (St. Aubin et al., 1986). The finding of these closed LMX elements far back from the tips of corn roots was a great surprise, because micrographs of carefully fixed and embedded tissues in these regions showed LMX elements with thick, lignified walls, open end walls, and contents (if any) dis- organized. Such preparations were taken as evidence of dead, functioning xylem conducting tubes. The reason soon became clear: The developing LMX elements in the grasses are very large cells (0.1 X 1.5 mm in corn). During usual preparative procedures, their end walls and cytoplasm are disrupted, but we found that the best way to preserve the end walls is with hand-cut sections of fresh tissue (Fig. 1C). Fixation before such sectioning also pre- served the cytoplasm (St. Aubin et al., 1986). In dicotyle-

dons the LMX is easily found by looking far enough back from the tip.

The discovery that the high-volume, low-resistance pipeline to carry transpiration water out of the roots was closed for 20 cm or more from the tip at once raised the puzzling question of where the water entered the roots. The answer given in all of the textbooks, that water enters in the region a few centimeters behind the tip, appeared not to be con- sistent with the absence of a pipeline to carry it. This question will be taken up below. Some of the reports of delayed xylem maturation are listed in Table I and include an early (unrecognized) report of immature LMX more than 1 m from the tip of banana roots. The development of conducting tubes in the secondary xylem of roots has received little attention, but a slow maturation of the com- ponent cells occurs in five herbaceous and two woody species studied recently (McCully, 1994).

One common feature of all roots is the long length over which an individual element develops from its small par- ent cell. Not only does the element enlarge for part of this distance but it often remains alive for a further long distance after lignification is complete (Fig. 3). This extended life of the large element is used in nutrient accumulation. High concentrations of potassium have been found by x-ray microanalysis in the immature LMX elements in roots of corn (McCully et al., 1987), barley (Huang and Van Steveninck, 19881, and soybean (McCully, 1994) and in the developing secondary xylem of all the dicotyledons ob- served (McCully, 1994). High turgor (0.6-0.8 MPa) has been measured with a pressure probe in slow-developing LMX elements of corn (Frensch and Hsiao, 1993). These observations have revived an old question: Are the solutes that enter the transpiration stream first loaded into living, immature xylem elements and then released into the tubes as each element cell dies rather than being loaded directly into the dead tubes? This question was raised initially by Hylmo in the 1950s and revived by Higinbotham and by

Figure 1. (On facing page) All except D and C show roots of field-grown corn. All except A are hand-cut sections of fresh material. Distances were measured from the root tip. A, Four-week-old plant freshly dug and shaken vigorously but not washed. X0.3. B, Mature region of a nodal root at approximately 50 cm. EMX (*) alternates with phloem poles. Two large, mature LMX elements have been sectioned transversely. Rhodamine B stain. Blue-light-induced fluorescence. X275. C, lntact end wall between two developing xylem elements in a nodal root at approximately 20 cm. Longitudinal section. Rhodamine B. Blue-induced fluorescence. X380. D, Main root of soil-grown soybean at 22 cm. The large, central LMX elements have thickened walls just beginning to lignify (blue stain). These cells were still alive, but preparation destroyed the cytoplasm. All EMX elements are lignified, and some of the youngest are still alive. Toluidine blue. Bright-field optics. X400. E, The rhizosheath on a young primary root at 20 cm. Dark field. X 15. F, Root cap of a nodal root, stained with neutra1 red and sectioned longitudinally through i t s periphery. The dye has accumulated in vacuoles of l iving cells. Mucilage produced by the peripheral cap cells has hydrated during the staining and pushed out the abscised cells and the soil clinging to them. These living cells and the mucilage become part of the rhizosheath, which forms back around the developing root hairs. X200. G, Periphery of a sugarcane root at approximately 20 cm. Most of the rhizosheath has been washed away during preparation. Soil still clings at anchor points at the curled regions of the root hairs. Acridine orange. UV-induced fluorescence. X100. H, Branch root with mature endodermis and a developing hypodermis already strongly suberized on the anticlinal and outer tangential walls. The epidermis with numerous root hairs is intact. Transverse section at approximately 1 mm from the tip that is becoming determinate. Periodic acid Schiff reaction followed by fluorol yellow. Blue-induced fluorescence. X225. I , Junction of young branch and main root. Cortex, hypodermis, and epidermis press against the branch to make a tight seal. The large, immature LMX in the branch adjoins phloem in the mother root. A bed of connecting xylem is developing in the main root stele. The LMX of the main root i s immature. Rhodamine B. UV-induced fluorescence. ~ 2 0 0 .

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McCully Plant Physiol. Vol. 109, 1995

Figure 2. Typical unwashed portions of field-grown corn roots. Cen-ter, Rapidly growing tip with rhizosheath formed behind the elon-gating region. Left, Older region with numerous branches also stillsheathed. Right, Mature region where the rhizosheath has been shed.Reprinted with permission (figure 2 of McCully and Canny11988]). XI.

Anderson and House almost two decades later, unjustlydiscredited shortly after, and debated very little ever since(for references and discussion, see McCully and Canny,1988). In contrast, the pathways and processes by whichnutrients and water enter roots and travel to the stele arestill hotly debated and much researched (Clarkson, 1993).

Where only the narrow tubes of the EMX are open forwater conduction, the suction from the shoot is sharplyattenuated and relatively little water will be pulled up fromthat portion of the root. Thus, the delayed development ofthe LMX must influence the water relations of the sur-rounding root tissue. In corn it has been shown that wherethe LMX is immature the relative water content (and water

Figure 3. A planed transverse face of a frozen soybean root at 22 cm.The central LMX is mature. The three developing conducting ele-ments of secondary xylem, all alive, have the highest K content in theroot. Reprinted with permission (figure 5 of McCully 11994]). X300.

potential) is much higher than in the older regions wherethe tubes are open (Wang et al., 1991). Furthermore, dailychanges of transpiration do not influence the steady, highwater content of the immature regions, but the regions withopen LMX dry dramatically during transpiration (Wang etal., 1991). One result of this isolation from the transpirationtension is that immature roots may conserve water inyoung and older seedlings until an extensive root system isestablished. Wenzel et al. (1989) showed that in corn noLMX is open in the roots of soil-grown seedlings up to asmany as 14 d after germination and that plants of corn andthree other C4 grasses as large as that shown in Figure 1Ahave as little as 14% and rarely more than 50% of total rootlength with open LMX conducting tubes. Another result ofthe closed LMX may be that the meristematic tissues areprotected from the transpiration tension.

DEVELOPMENT OF THE INTERFACE WITH THE SOIL

The freshly excavated root system of a young corn plant(Fig. 1A) is seen to have the oldest roots (here the seminal

Table I. Vessel maturation in roots of several speciesDistances from the root tip at which EMX and LMX conducting vessels become open tubes. -, Not

recorded.

Plant Type of RootDistance from Tip

ReferenceEMX LMX

Banana

CornBarleyBarleySoybeanWheat

a Peripherallignification.

Nodal

NodalSeminalSeminalMainSeminal

LMX. b Central

___---

16.3c'f10d.f

6.5°-'

LMX.

cm

20-40a

60-1 30b

20-30+1520

17 +28.8c'f

23.8dJ

11.3eJ

cAt 10°C.

Riopel and Sleeves (1964)

St. Aubin et al. (1986)Huang and Van Steveninck (1988)Sanderson et al. (1988)Kevekordes et al. (1988)Huang et al. (1991)

d At 20°C. ° At 30°C. ' First



Some New Views of Root Structure

roots) free of soil, whereas the younger nodal roots aresheathed in soil (rhizosheath), except just behind the tip. Atfirst we thought that there were two types of root, bare andsheathed (Vermeer and McCully, 1982); we now under-stand that these are stages of a developmental process thatoccurs along each root (Fig. 2). The root tip is bare in theelongation region and where root hairs develop. The soilsheath extends over that portion of the root that retainsliving root hairs and epidermal cells. As branch roots de-velop in the sheathed region they also become sheathed.The sheathed regions of rapidly growing main roots extendin corn 20 to 30 cm or more along the root. Beyond this thesheath is lost, the main root becomes bare, and the epider-mal cells die and disintegrate.

The central, illuminating fact that has united all of theseinvestigations is that a soil sheath outside the root is onlypresent where immature, nonconducting LMXs are inside.This was discovered first for corn (St. Aubin et al., 1986)but is true for a wide range of grasses examined subse-quently (McCully and Canny, 1988). The discovery of thisfact has enabled us to bring together the water relations ofthe root with the processes in the soil at the root surface.

The rhizosheath is a feature of grasses and is found on allmesophytic cereals (Vermeer and McCully, 1982; Duell andPeacock, 1985; McCully and Canny, 1988). It is a coherentstructure (Fig. IE), in corn about 2.5 to 3 times the volume ofthe subtending root (in small grains like barley up to 16times). It is tightly bound to parts of the root hairs. Its outersurface is discrete and separates cleanly from the surroundingsoil. Rhizosheath development is the result of an interplaybetween the plant, the soil, and rhizobacteria, the complexi-ties of which we are just beginning to appreciate. We nowknow that the aggregation of soil particles in the rhizosheathsis caused partly by the mucilage released from the root cap,

Figure 4. Root-soil interface in the rhizosheath of barley, frozen at 7AM. The root hair and epidermis are in close contact with soil andexpanded mucilage. E, Epidermis. Cryoscanning EM. Reprinted withpermission (figure 3D of McCully [1995]). X730.

together with cap cells that remain alive all along the rhizo-sheath (Vermeer and McCully, 1982; Watt et al., 1994). Theseroot-cap products, formed as long as the apical meristemremains active (Fig. IF), are left behind as the root extends.They do not stick to the first centimeter or so of the root,which is covered by a special temporary pellicle layer (Abey-sekera and McCully, 1993), but they lodge in the soil and roothair zone. This cap-derived mucilage, together with muci-lages from bacteria specific to this region of the root, help toglue the soil to the root (Watt et al., 1994). Root hairs becomemuch distorted in the sheath-forming region, and the soil isanchored at these distortions (Fig. 1G), where characteristicassociated bacteria flourish (McCully and Canny, 1988). Soilmoisture also influences the rhizosheath, a much heavier,more coherent sheath developing under dry conditions (Wattet al., 1994).

Plants with rhizosheaths have an amplified root surfacein intimate contact with a considerable volume of soil. In acorn plant like that in Figure 1A, this would be approxi-mately 80% of the root length (Wenzel et al., 1989). Al-though there are spaces in the rhizosheaths (at least whenplants are transpiring), parts of each root hair are always incontact with soil, and when plants are guttating hairs maybe surrounded by mucilage (Fig. 4) expanded by exudedwater (McCully, 1995). This must facilitate water and ionuptake, particularly in dry soils. Corn nodal roots areknown to shrink in diameter under water stress, and it hasbeen assumed that this shrinkage would create a gap,greatly increasing the resistance for water movement fromsoil to root (Nye, 1992). But shrinkage of the root portionunderlying a rhizosheath would draw the sheath with it.Separation from the bulk soil would occur at the outersurface of the sheath. Thus, in the sheathed region therewould be a buffer of attached soil from which the rootcould continue to extract nutrients and some water.

Uren (1993) has proposed that the penetration of rhizo-sphere mucilages into small pores between soil particles ef-fects release of manganese and possibly iron by a process ofcontact reduction, a process that would be aided by the highmucilage content and close packing of the soil within rhizos-heaths. The rhizosheath is probably wetter than the soil far-ther away, because it is attached to the wet zone of the root.It therefore provides a unique environment for soil microor-ganisms. Gochnauer et al. (1989) have shown that the popu-lation of bacteria in the rhizosheath of corn is distinct fromthat associated with the older, unsheathed regions. There hasbeen no experimental work on whether roots that do not formrhizosheaths (most of the dicotyledons) also have high waterstatus in the zones where the LMX conducting tubes areimmature, nor is it known whether these root zones haveunique surface properties, characteristic bacteria, or effects onrhizosphere development.

BRANCH ROOTS

The branch roots in corn provide a possible solution tothe puzzle of why the high-capacity pipeline of the mainroot LMX does not open until far back from the tip. Thebranches collect 80% of the water entering a large rootsystem (Varney and Canny, 1993). At first glance they www.plantphysiol.orgon September 12, 2020 - Published by Downloaded from



6 McCully Plant Physiol. Vol. 109, 1995

appear to be poor candidates for this role, because they are mostly short (mean 3 cm), and those where the main root LMX is open have always lost their apical meristem and have the apoplastic barriers of both endodermis a n d hypodermis mature (Fig. 1H) right to their tips (Wang et al., 1995). However, the main root LMX becomes mature a t the place where the branch LMX tubes open to supply them, a n d the collecting surface for water uptake increases 8-fold. Unexpectedly, the epidermis of even old branches remains alive, thus facilitating the uptake of water and nutrients into the symplast (Wang et al., 1995). Branch roots have extensive and complex vascular connections with main roots (Fig. 11). At these junctions, vessels and sieve tubes lie side by side (McCully and Mallett, 1993). These may be the bridges where carbon and nitrogen compounds from the root recycle back to the shoot. Thus, the branch roots, largely unstudied until now, hold many clues to the work- ing of a real root system.

PROSPECTS

The way plants work can be understood only i n terms of the way they are p u t together, a s I have tried to illustrate i n this article. The structure cannot be taken for granted: root systems are not just multiples of seedling roots. Field- grown roots have features unknown from solution culture. Each plant a n d each situation must be examined.

So far, most of the structural/functional investigations of field-grown roots have been done on only a few species, with emphasis on the grasses. A n d the grasses have been particularly revealing because of their rhizosheath development, which has led to the findings of the delayed development of the xylem a n d of the far-reaching effects on the water and nutrient relations of the different parts of the root system. W e also begin to see outlines of the obviously much larger role played i n the development and function of the associated rhizosphere soil by the heterogeneity and developmental sequences within a root system.

Much remains to be discovered by continuing studies of the grasses. The herbaceous dicotyledons and the trees are still almost untouched, b u t similar studies of them will certainly also bring great rewards and even more surprises about how real roots work.

ACKNOWLEDGMENTS

Thanks to Michelle Watt and Xue-Lian Wang for Figure 1, E and H, respectively.

Received March 8, 1995; accepted April 20, 1995. Copyright Clearance Center: 0032-0889/95/109/0001/06.

LITERATURE ClTED

Abeysekera RM, McCully ME (1993) The epidermal surface of the maize root tip. I. Development in normal roots. New Phytoll25: 41 3-429

Clarkson DT (1993) Roots and the delivery of solutes to the xylem. Philos Trans R Soc Lond Biol Sci 341: 5-17

Dittmer HJ (1937) A quantitative study of the roots and root hairs of a winter rye plant (Secale cereale). Am J Bot 2 4 417-419

Duell RW, Peacock GR (1985) Rhizosheaths on mesophytic grasses. Crop Sci 25: 880-883

Frensch J, Hsiao C (1993) Hydraulic propagation of pressure along immature and mature xylem vessels of roots of Zea mays measured by pressure-probe techniques. Planta 190: 263-270

Gochnauer MB, McCully ME, Labbé H (1989) Different popula- tions of bacteria associated with sheathed and bare regions of roots of field-grown maize. Plant Soil 114: 107-120

Huang B, Taylor HM, McMichael BL (1991) Effects of tempera- ture on the development of metaxylem in primary wheat roots and its hydraulic consequences. Ann Bot 67: 163-166

Huang CX, Van Steveninck RFM (1988) Effect of moderate salin- ity on patterns of potassium, sodium and chloride accumulation in cells near the root tip of barley. Role of differentiatmg metaxylem vessels. Plant Physiol 73: 525-533

Kevekordes KG, McCully ME, Canny MJ (1988) Late maturation of large metaxylem vessels in soybean roots: significance for water and nutrient supply to the shoot. Ann Bot 62: 105-117

McCully ME (1994) Accumulation of high levels of potassium in the developing xylem elements in roots of soybean and some other dicotyledons. Protoplasma 183: 116-125

McCully ME (1995) Water effux from the surface of field-grown roots. Observations by cryoscanning electron microscopy. Physiol Plant (in press)

McCully ME, Canny MJ (1988) Pathways and processes of water and nutrient uptake in roots. Plant Soil 111: 159-170

McCully ME, Canny MJ, Van Steveninck RFM (1987) Accumu- lation of potassium by differentiating metaxylem elements of maize roots. Physiol Plant 69: 73-80

McCully ME, Mallett J (1993) The branch roots of Zea. 111. Vascu- lar connections and bridges for nutrient recycling. Ann Bot 71: 327-341

Nye PH (1992) Towards the quantitative control of crop produc- tion and quality. 111. Some recent developments in research into the root-soil interface. J Plant Nutr 1 5 1175-1192

Riopel JL, Steeves TA (1964) Studies on the roots of Musa acumi- nata cv. Gros Michel. I. The anatomy and development of main roots. Ann Bot 28: 475489

Sanderson J, Whitbread JC, Clarkson DT (1988) Persistent xylem cross-walls reduce the axial hydraulic conductivity in the apical 20 cm of barley seminal root axes: implications for the driving force for water movement. Plant Cell Environ 11: 247-256

St. Aubin G, Canny MJ, McCully ME (1986) Living vessel elements in the late metaxylem of sheathed maize roots. Ann Bot

Uren NC (1993) Mucilage secretion and its interaction with soil, and contact reduction. Plant Soil 155/156: 79-82

Varney GT, Canny MJ (1993) Rates of water uptake into the mature root system of maize plants. New Phytol 123: 775-786

Vermeer J, McCully ME (1982) The rhizosphere in Zea: new insight into its structure and development. Planta 156 45-61

Waisel Y, Eshel A, Kalkafi U, eds (1991) Plant Roots: The Hidden Half. Marcel Decker, New York

Wang XL, Canny MJ, McCully ME (1991) The water status of the roots of soil-grown maize in relation to the maturity of their xylem. Physiol Plant 8 2 157-162

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5 8 577-588



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