Plant Soil (2009) 322:25–37 DOI 10.1007/s11104-008-9861-4

REGULAR ARTICLE

Shovel roots: a unique stress-avoiding developmental strategy of the legume coronarium L.

Elisabetta Tola & Josè Liberato Henriquez-Sabà & Elisa Polone & Frank B. Dazzo & Giuseppe Concheri & Sergio Casella & Andrea Squartini

Received: 22 October 2008 /Accepted: 10 December 2008 /Published online: 21 January 2009 # Springer Science + Business Media B.V. 2009

Abstract Hedysarum coronarium () is a legume accumulate this cation intracellularly as insoluble native to the Mediterranean basin, known for its broad crystalline salts. Such bioaccumulation results in a tolerance to various environmental stresses, and its localized depletion of CaCO3 from the soil. As a ability to thrive without signs of chlorosis when consequence of this removal of the pivotal carbonate growing in arid and alkaline soils up to pH 9.6. A buffering system, the iron-solubilizing acidification unique but poorly known morphological feature of its activities of the roots can become effective. Further root system is the production of “shovels”, modified tests revealed that the factor triggering shovel lateral roots that acquire a curved and flattened shape. development is exposure of roots to iron oxide. This A combined structural and functional analysis was signal, reporting at once both iron presence and undertaken to define the nature and role of the shovel alkalinity, assures the availability of iron nutrient roots using various microscopy techniques, histo- reserves upon acidification of the local microenvi- chemical stains, STEM - energy dispersive X-ray ronment surrounding the roots. These findings, microanalysis, infrared spectroscopy, and plant culti- besides casting light on a novel and unique botanical vation in different conditions. We found that sulla phenomenon, offer the potential to exploit sulla’s displays remarkable unique rhizosphere-buffering model and genes for the improvement of other crops properties at both ends of the pH scale, and that to sustain productivity in a scenario of climate shovels act as efficient calcium-absorbing organs that warming and increasing desertification.

Keywords Hedysarum coronarium . . Responsible Editor: Hans Lambers. Shovel roots . Calcium crystals . Iron nutrition . E. Tola : J. L. Henriquez-Sabà : E. Polone : G. Concheri : Calcareous soil S. Casella : A. Squartini (*) Dipartimento di Biotecnologie Agrarie, Università di Padova, Introduction Viale dellUniversità 16, 35020 Legnaro, Padova, Italy e-mail: [email protected] The perennial legume Hedysarum coronarium L. (originally included in the ), is a F. B. Dazzo member of the tribe , subtribe Euhedysar- Dept. of Microbiology and Molecular Genetics, Michigan State University, inae whose natural range in the Mediterranean basin East Lansing, MI 48824, USA stretches from Morocco, Algeria, and Tunisia to 26 Plant Soil (2009) 322:25–37 southern , Balearic Islands, Corsica and central mycorrhizal fungi, their contribution to plant growth to southern Italy. This plant is known under the and P content, for some fungal as Glomus vernacular names of sulla, Spanish sainfoin, and caledonium, can be unusually low or even detrimental Spanish esparcet. A separation from the genus (Lioi and Giovannetti 1987). The plant mineral Hedysarum and the constitution of the new genus content also denotes an uncommon efficiency for Sulla has been proposed (Choi and Ohashi 2003)and iron uptake as this element constantly amounts to in recent literature the plant has been also referred to values above 300 mg/kg dry weight throughout all as Sulla coronaria. Besides its native occurrence in vegetative stages (Leto et al. 1989) while the mean calcareous pliocenic clays, marnous slopes and reported for legume forages is 222 mg/kg and that for desert soils, sulla is well exploited as a forage crop grass forages is 184 mg/kg (Spears 1994). Such a due to its pronounced drought resistance, strong difference is remarkable also in light of the fact that it tolerance to alkaline soils (Lupi et al. 1988), good is achieved by sulla equally well in neutral and agronomical yield (up to 60 tons of green forage per alkaline soils where iron solubility is at its lowest. We hectare per year), and protein content (up to 24% of have previously characterized various aspects of leaf dry weight) (Ballatore 1963; Sarno and Stringi sulla’s biology, including the histological details of 1981). It has also been successfully tested for its microbial interactions (Squartini et al. 1993) from cultivation in California (Mitchell et al. 1999)and which we subsequently described and named its Australia (Casella et al. 1984). nitrogen-fixing bacterial endosymbiont Rhizobium Sulla thrives in alkaline calcareous clay soils at a sullae as a new species in bacterial pH up to 9.6, which represents a definite extreme in (Squartini et al. 2002). In the present work we address plant physiology, and is also beneficial in arid zones a morpho-physiological peculiarity, so far observed due to its anti-erosion properties conferred by a only for species of Hedysarum, which under certain vigorous root system. Figure 1 shows the plant soil conditions develops modified flattened and curled growing profusely in one of its extreme habitats that lateral roots. These unusual root organs had been are prohibitive to other vegetation. This plant is also noted on H. coronarium in the early 1800’s by classic well suited to grow in soils containing low amounts of botanists such as De Candolle, and were named bioavailable phosphorus, and, uncommonly for a “palette” (plural of paletta, Italian for “shovel”)by legume, does not consistently rely on mycorrhizal Mottareale (1898) due to their flat and curved shape. infection to assist in sequestering this mineral nutri- A century ago Severini (1908), following field ent; in fact, although roots can be colonized by observations and accounts from farmers, reported that

Fig. 1 Hedysarum coronar- ium (sulla) showing profuse growth in a bare pre-sahar- ian area (Northern Algeria). The dark green foliage indicates a fully efficient mineral nutrition. Some sat- ellite vegetation (crucifer family) is visible within the sulla bush benefiting from its effects Plant Soil (2009) 322:25–37 27 they resembled “leafy and fragile” lateral roots that 0.12 g/l MgSO4·7 H2O, 0.1 g/l KH2PO4,0.15g/l appear to accumulate limestone, eventually become Na2HPO4·2 H2O, 0.005 g/l Fe citrate, 0.1 mg/l H3BO3, salt-encrusted and spontaneously detach from the 0.1 mg/l MnCl2,0.1mg/lNa2Mo4O4,0.1mg/lZnCl2, plant. Both these early reports appeared only in 0.1 mg/l CuSO4, pH 6.5. To determine if bacteria Italian in local bulletins. Recently we have also influence shovel development, germinated seedling roots observed the same structures in root systems of other were inoculated by dipping into 20 ml of Fahräeus wild Hedysarum species such as H. glomeratum and medium containing a suspension (107 cells/ml)ofthe H. spinosissimum, which are taxonomically close Rhizobium sullae microsymbiont (Squartini et al. 2002) relatives of H. coronarium and occupy the same grown for 5 days on BIII agar (Dazzo 1982). To test how habitat. Despite their long-standing records of obser- varying the dosage of Ca + + influences shovel vation, the so-called shovels have not received a development, CaCl2 at 0.0, 0.1, 0.5, 1.0 and 5.0 g/l was thorough scientific investigation. Thus, the focus of supplemented in the medium. Alternatively a modified this work was to investigate their formation, structure Leonard’s solution was used (0.174 g/l KH2PO4, and function using stereomicroscopy, brightfield, 0.34 g/l CaSO4·2H2O, 0.174 g/l MgSO4·7 H2O, phase contrast and polarized light microscopy, histo- 0.170 g/l KCl, 0.07 g/l Fe citrate, 0.04 mg/l CuSO4·5H2O, chemical stains, STEM - energy dispersive X-ray 0.1 mg/l ZnSO4·5H2O, 1.0 mg/l MnSO4·1H2O, microanalysis, infrared spectroscopy, along with 0.05 mg/l (NH4)2MoO4·1H2O, 0.7 mg/l H3BO3,pH hydroponic and field-level plant cultivation under 6.8. This medium was further modified to test for various environmental conditions. effects of varying the dosage of K2HPO4 (from 2× to 30×), of CaSO4·2H2O (from 2× to 30×), and of ferric citrate (from 2× to 10×). Ferric chloride at Materials and methods concentrations of 0.07–1.00 g/l was also tested as a substitute for ferric citrate. To test whether Fe0 and Plant growth conditions iron oxides affected shovel development, solid metallic iron bars were submerged in the bottom Natural were collected in May–June from their reservoir. No forced aeration was used in the habitat (pliocenic clays of Villamagna and Volterra in systems. All the above tests were run using five the Tuscany region of Italy). For parallel laboratory replicate plants. Plants were grown in a growth cultivation, Hedysarum coronarium seeds were sur- chamber illuminated with mixed incandescent/fluo- face-sterilized by immersion in absolute ethanol for rescent lighting (ca. 400 μE/m2/sec) and programmed

30 s, then transferred into 0.1% HgCl2 and stirred for with a 16 h photoperiod, 22°C/20°C day/night cycle, 7 min, and finally rinsed in seven changes of sterile and 70% relative humidity. The liquid in the bottom deionized water. Seeds were imbibed for 3 h in the reservoir of the Leonard Jar was replaced once during final wash. Seeds were placed on inverted TY agar the growth period and plants were harvested 45 days (Beringer 1974) Petri plates, wetted with ten drops of after germination. sterile water, and incubated 3 days in the dark to In experiments aimed at measuring the pH shift of germinate. The growth system used for these studies the growth medium by seedling roots, 5-day old was the Leonard jar (Leonard 1943), where the tested germinated plants were placed on top of pierced caps solution was delivered to the plant roots by capillary of scintillation counter glass vials with their roots flow through a cotton gauze wick into the rooting penetrating into the vials that contained unbuffered medium substrate in which the plants were lodged. sterilized Fahräeus solution prepared with 0.1 g/l KCl

Tests were run using sterilized media in order to avoid instead of KH2PO4 or twice the amount of Na2HPO4 microbe-mediated effects. Axenic seedlings were (0.3 g/l). The solution was supplemented with the transferred aseptically into sterilized plastic Leonard Carlo Erba Liquid Chromogenic pH Indicator (Carlo jars filled with a 1/1 mixture of water-washed, oven Erba Reagenti, Rodano, Italy), active in the range of dried, perlite and vermiculite, and fed from the pH 1–11. Seedlings were incubated for 3 days in the bottom with sterile nitrogen-free Fahräeus solution growth cabinet at the above conditions, and the pH of (Fahräeus 1957) a medium commonly used for the rooting medium was monitored daily by visually hydroponical purposes containing: 0.1 g/l CaCl2, comparing its solution color to the reference scale of 28 Plant Soil (2009) 322:25–37 the pH indicator. As a control plant, Trifolium repens (Phaseolus vulgaris) were used as a positive control (white clover) Cv. Dutch White was compared to for this cytochemical staining of calcium oxalate sulla. Experiments were run in triplicates. crystals. The test for calcium carbonate was carried out by excising crystals from mature shovels with a Microscopy and energy dispersive x-ray scalpel and forceps, placing them in 2% HCl and microanalysis inspecting for liberation of gas bubbles during crystal dissolution under a dissecting microscope. A total of 30 plants grown hydroponically in laboratory conditions and a corresponding number Infrared spectroscopy harvested from their natural habitats were analyzed. Root structures were examined by various types of IR spectra of isolated, dry crystals excised with a microscopy. Stereomicroscopy was used to docu- scalpel blade from mature shovels and freed from ment the general structure of shovels on intact roots. organic debris, were recorded on a Perkin–Elmer FT- To reveal the vascular system of shovels, roots were IR Model 1760X spectrometer. The spectra were cleared with diluted sodium hypochlorite, stained obtained at room temperature after grinding crystals briefly with methylene blue solution and examined into a mini agate mortar for 10 min. One hundred by brightfield microscopy as described (Philip- scans of 2 cm −1 resolution were acquired from the Hollingsworth et al. 1991). For more detailed ground material, signal-averaged and stored on a histological and cytological examinations, shovel magnetic disk. tissues were fixed and embedded as described by

Subba-Rao et al. (1995)exceptthatCaCl2 was omitted from the fixation steps. Light microscopy Results was done on 2–3 μm sections stained with alkaline toluidine blue, followed by computer-assisted digital 1) Occurrence of shovels in nature and in laborato- morphometry (Dazzo and Petersen 1989)usingthe ry-grown plants Bioquant System IV Image Analysis software. To detect crystals within shovel tissue, 2–3 μmthick When examining wild plants of Hedysarum coro- longitudinal sections were examined by phase narium growing in their natural habitat in the Tuscany contrast microscopy and polarized light microscopy calcareous hillsides, over 95% of the root systems using crossed polarizers. For elemental analysis, the observed in 30 plants bore shovels, whose number embedding and fixation method of Subba-Rao et al. could vary from a minimum of one per plant to over (1995) was used, and ultrathin (100 nm thick) 120 in occasionally crowded and stacked roots. The sections were prepared using a diamond knife. mean number was 7.8±1.6 SE per plant. When we Sections were carbon-coated and examined with a grew 30 plants in hydroponic conditions in Leonard JEOL 100CX-II scanning-transmission electron mi- jars watered with Fahräeus medium shovels were croscope operating at 80 KV and equipped with a formed in 52% of the cases and in these the mean was Link x-ray microanalysis system. The preparation 3.2±0.6 SE shovels per plant. ensures that once under the microscope, the elements In comparison with the number of normal lateral in the sections were only those retained after the roots, shovels were always a minority accounting for fixation and embedment solutions. Selected area 2–4% of the total branches of the parent root with no analysis was performed at 60,000 X with counts significant differences between field- and laboratory- collected at 200 s intervals. grown plants. 2) General morphology Cytochemical tests Shovels develop various morphologies on the root Mature, crystal-containing shovels were treated with the system of Hedysarum coronarium. The most common rubeanic acid cytochemical stain for calcium oxalate one is a laterally branched, short, flattened shovel- deposits (Yasue 1969; Horner and Zindler-Frank 1982). shaped structure with a slight geotropic curvature Leaves of freshly collected field-grown common bean (Fig. 2a, left root) that differs from the typical aspect Plant Soil (2009) 322:25–37 29

Fig. 2 Roots of H. coro- narium plants grown in na- ture presenting a variety of shovel shapes. a left: flat and concave leafy-style ma- ture shovels, compared with root nodules, (right), b ex- ample of high density stack of shovels; c pleomorphism of shovels elongated into flexible ribbons (indicated by arrows). Scale bars: 3 mm in a and 10 mm in b and c. (Modified from Squartini et al. 1993). For the appearance of regular lateral roots see also the left plant in Fig. 9

of nitrogen-fixing nodules induced by the symbiotic 3) Abundant root hairs and small cells are present on bacterium Rhizobium sullae on the same plant the concave side of the shovels (Fig. 2a, right root). Unlike root nodules, shovels can develop under axenic hydroponic culture in Incident light stereomicroscopy further revealed Fahräeus medium, thus ruling out a microbial distinctive cytological features of shovels (Fig. 4). In requirement for their induction. The three shovels in all shovels analyzed, consisting in over 60 specimens, Fig. 2a also illustrate the typical fragile state of these root hairs developed profusely on the underside of the organs at maturity due to the copious accumulation of shovel (Fig. 4, bottom), suggesting that this organ has crystalline deposits. Shovels can vary in length, width efficient absorbing functions. and density on the root, sometimes forming thick, Cross-sections of shovels (Fig. 5a) revealed their stacked piles (Fig. 2b) or elongated and twisted overall ellipsoid shape, a greater abundance of root ribbons (arrows in Fig. 2c). Their shape and firmness hairs on their underside, an expanded cortex and the vary with soil texture, being short and stiff when near-central location of their vascular tissue. The size grown in compact limestone-rich ground, and long and flexible when grown on sandy substrates or in hydroponic culture. Their number and density depend on the level of inducing conditions as described below. While regular lateral roots appear indetermi- nate in growth, shovels are generally much shorter when compared to normal branches of the same age and order. The histology of vascular tissues does not show differences from that of regular roots. Brightfield microscopy of shovels cleared with sodium hypochlorite to visualize their internal histol- ogy revealed a central (rather than peripheral) vascular system and expanded cortex (Fig. 3), indi- cating that they were short, thick lateral roots with Fig. 3 Brightfield microscopy observation of a shovel after sodium hypochlorite root bleaching of the root. A central histological features that distinguish them from the vascular system is evident. Plants were grown in Fahräeus bacteria-induced root nodules. medium for 3 weeks. Scale bar equals 0.2 mm 30 Plant Soil (2009) 322:25–37

c). This type of microscopy enables only the light from anisotropic, ordered structures (e.g. cellulose fibers and micelles, crystals etc.) to pass while blocking light transmitted by amorphous material. The results showed that those intracellular granules were indeed crystalline in nature, indicating that shovels are sites of crystal accumulation. Eventually the crystals grow to macroscopic size (1–4 mm long). At maturity the whole shovel is mineralized, can easily crack, detach from the root and remain in the soil. Tilled soils in Tuscany after a cultural cycle of sulla reveal numerous typical remains of these mineralized shovel structures. Large crystals that are millimeters in length can be easily extracted by manual dissection of mature shovels.

Fig. 4 Incident light steromicroscopy view of shovels on primary roots of H. coronarium. young stages of growth (top). Elongated shovel starting to curl (bottom); the plants were grown in Fahräeus medium as described. Root hairs are visible in the concave side. Scale bars equal 2 mm of individual cortical cells in cross-section is larger when located above the central vascular system (Fig. 5a). Figure 5b shows this difference in distribu- tion of the cross-sectional area of cortical cells above and below the central vascular system analysed by digital morphometry. Data from ten mature shovels were analyzed upon cross-sectioning the shovel at its point of maximum width, which was normally located near the middle of its length. The asymmetric distribution in cortical cell dimension suggests how elongated shovels acquire their slight geotropic curvature (Fig. 4, bottom). The uneven cell size forces the growing structure to curve its axis and explains the final curled shovel-like shape. 4) Shovels progressively accumulate crystals of calcium salts Fig. 5 a light microscopy observation of a shovel’s cross- section. The isolated small cells on the bottom are sectioned Phase contrast light microscopy of longitudinal root hairs. Scale bar equals 0.2 mm. b BioQuant digitized morphometrical assessment of the cell size distribution for the sections of shovels from 4 week-old plants indicated upper (non-root hair side) vs. lower (root hair side) longitudi- multiple deposits of intracellular refractile granules nal portions of Fig. 5a. The Y axis reports the prercentage (Fig. 6a). Such an appearance is also compatible with values in which each of the cell size classes of the X axis are phenolic droplets which can occur in various plant distributed. Processing the image with computer-assisted digitized morphometry quantifies the larger average cross tissues. Cross-polarized light-microscopy was done to sectional area of the cells residing in the upper side of a middle determine if these granules were crystalline (Fig. 6b, line drawn slightly above the stele Plant Soil (2009) 322:25–37 31

Fig. 6 a Phase contrast light microscopy on toluidine blue- same section observed with crossed polarizers, showing only stained longitudinal sections of a young shovel (from a objects of ordered structure. Cell walls (orange-red) are 45 days-old plant) showing refractile bodies included within partially bright due to their semi-anisotropic nature, while cells. Three of the several corpuscles of such type are indicated crystal bodies inside cells fully transmit polarized light and by the arrows. b Cross-polarized light microscopy on a appear as bright white spots. Scale bar is 50 μm in (a) and sectioned 4-week old shovel, with parallel polarizers. c The 400 μm in (b) and (c)

Several approaches were used to define the tested negative with this cytochemical stain, in chemical nature of these crystals. Scanning/trans- contrast to positive staining controls of calcium mission electron microscopy (STEM) of sections oxalate within mature bean leaves (not shown). made of shovels on plants grown in Fahräeus The alternative possibility that these shovel crystals medium indicated various sized, electron opaque might consist of calcium carbonate was tested by intracellular bodies (Fig. 7b). Energy dispersive x- application of 2% HCl that would cause them to ray microanalysis of these granules indicated that they dissolve accompanied by conspicuous liberation of contained calcium (Fig. 7a), plus other cations (Cu CO2 bubbles which was observed visually under a being not indicative in this technique as it originates stereomicroscope. Interestingly, crystals obtained from from the supportive copper grid). Regarding anions, shovels on H. coronarium plants growing in the phosphorus was definitely abundant in the x-ray calcareous alkaline pliocenic clay soil of Tuscany fluorescence spectrum and a likely candidate. How- tested positive for calcium carbonate with both ever, this method is unable to detect carbon, due to its solubilization and gas formation, whereas shovel mass and the abundant background of formvar coating crystals from plants that had been grown in sand of the grids. Therefore, in order to define whether the watered with Fahräeus solution in the laboratory, crystals are made of calcium phosphate, calcium dissolved without effervescence. The former result carbonate or other calcium salts, the following com- would therefore be consistent with a limestone (calci- plementary techniques were used. um carbonate) composition, in agreement with the Some plants are reported to accumulate crystals of early hypotheses of Severini (1908), while the latter calcium oxalate (Arnott and Pautard 1970; Wu and result (dissolving without effervescence) more likely Kuo 1997) that can be detected in planta using indicated that crystals were made of calcium phos- rubeanic acid as a specific cytochemical stain (Yasue phate. These dissimilar results and their interpretations 1969). Legumes accumulate calcium oxalate within were resolved by infrared spectroscopy of crystals root nodules (Sutherland and Sprent 1984). However, isolated from shovels on plants developed under the crystals from shovels of H. coronarium plants always two different conditions. Shovel crystals formed by 32 Plant Soil (2009) 322:25–37

with pH values as high as 9 (Severini 1908; Sarno and Stringi 1981, Nuti and Casella 1989). Our studies indicated good vegetative growth and root nodulation by symbiotic Rhizobium bacteria in washed potted sand or in Leonard jars irrigated with Fahräeus solution adjusted to pH values up to 9.6. Since the bacterial symbiont Rhizobium sullae itself is tolerant only up to pH 8.7 (Struffi et al. 1998), the plant is likely performing rhizosphere-buffering strategies. In order to test this hypothesis, we grew seedlings of H. coronarium in a hydroponic system with unbuffered Fahräeus medium adjusted to different initial pH values, and recorded the change in pH after 3 days of growth using a non-intrusive system based on a color-changing soluble pH indicator active in the range between pH 1 and 11. For comparison, the forage legume white clover (Trifolium repens) was used as a control plant. The results (Table 1) show that H. coronarium is capable of nearly neutralizing the pH of its external rooting medium when its starting pH is between 6 and Fig. 7 Energy dispersive x-ray elemental microanalysis and 9. On the contrary, white clover was able to neutralize STEM electron microscopy of a 45 days old shovel: a elemental spectrogram from a scan of the irregular electron a slightly acidic rooting medium but totally unable to dense body (indicated by the arrow in Fig 7b) showing neutralize the medium from any of the three initial prominent presence of calcium and phosphorus (copper is from alkalinities tested. the microscopy grids). b corresponding STEM micrograph. The round-shaped electron-dense particles associated with vacuoles 6) The key condition inducing shovel formation is are presumably phenolic deposits, while the elongated structure exposure to iron oxide (arrow), compatible with the nature of a growing crystal, is the object of the x-ray elemental scan. Scale bar equals 25 μm We have observed that H. coronarium shovel plants growing in the Tuscan alkaline soils (Fig. 8a), formation either in nature or in laboratory can be which are a prime habitat for H. coronarium, had an IR neither regular nor homogeneous, varying from zero spectrum consistent with that of CaCO3 (Fig. 8c). In to several dozens per root apparatus, apparently contrast, shovel crystals isolated from laboratory- depending on the soil chemistry and substrate grown plants raised in sand and watered with Fahräeus composition. In order to clarify which situation is solution (Fig. 8b) had spectra more similar to those of most conducive to the development of this phenotype calcium phosphates (Fig. 8d). Neither of these shovel we carried out trials in defined media testing different crystal IR spectra matched that of calcium oxalate compounds and conditions. The growth condition (Fig. 8e), confirming the negative cytochemical test used for these studies was the Leonard jar system; obtained using the rubeanic acid stain. The large variations in the growth medium that were tested granular crystals that can be easily dissected from included different start pH conditions, excess or mature dry shovels are shown in Fig. 8f. deficiency of calcium, phosphorus, different forms of iron or other elements. Among the factors tested, in 5) Hedysarum coronarium, unlike other plants, can triplicate replicates, only the presence of oxidized iron adjust its rhizosphere to a wide range of acidic produced a clear-cut response triggering a massive and alkaline pHs production of shovels. When H. coronarium plants were grown with a source of iron prone to oxidation Sulla, as mentioned earlier, is renowned for its (e.g., metallic iron bars that rusted after few days), the remarkable unique ability to grow in alkaline soils plants developed many shovels. A mean of 49 shovels Plant Soil (2009) 322:25–37 33 Fig. 8 Infrared spectra obtained from crystals of mature shovels from plants grown as indicated (a,b), compared with those of pure compounds (c,d,e). Panel f shows examples of crystals (indicated by the arrow) erupting from drying mature shovels from plants grown in sand with Fahräeus me- dium. Scale bars in panel f equal 2 mm in the top images and 1 mm in the bottom images

per plant (±4.0 SD) were recorded, while any of the covered with brownish rust particles and developed other treatments, including the control plain Leonard an abundance of shovels on its roots. In contrast, solution, gave a mean not exceeding 0.6 shovels per other iron sources such as ferric citrate from 0.07– plant (±1.57 SD). Figure 9 shows a control plant with 0.7 g/l or ferric chloride from 0.07–1 g/l, were not no shovels (left) and a plant grown in the presence of effective in inducing shovel formation at various iron oxide (right), which, as a consequence, was concentrations tested, showing that a precipitated iron form was necessary to trigger the phenomenon. Table 1 pH of unbuffered Fahräeus medium after supporting Timewise, iron oxidation preceded shovel formation. 3 days of seedling root growth. Five replicates were used for This finding prompted our proposal of a model that each pH value. Mean pH ± SE are shown explains why shovels develop abundantly in soils Initial pH Final pH after 3 days of growth leading to iron precipitation and how their localized removal of calcium may counteract such limitation. H. coronarium T. repens

5.50 6.50±0.08 6.75±0.06 6.00 6.12±0.05 7.50±0.11 Discussion 7.00 6.75±0.09 7.00±0.05 7.50 7.00±0.07 7.50±0.09 The following aspects emerge from this analysis. (1) 8.00 7.20±0.12 8.00±0.04 The shovel root phenotype, currently known to be 9.00 7.40±0.21 9.00±0.05 uniquely expressed in the genus Hedysarum, develops 34 Plant Soil (2009) 322:25–37 Fig. 9 root apparati of Hedysarum coronarium plants grown in Leonard jars without (left)orwith(right) a source of iron oxide. Scale bar equals 1.5 cm

independent of microbial presence or signals made by inward side of such flat and curved roots. The them, and is represented as modified lateral roots with cupping of the shovel roots thus results in a greater a central vascular system. By comparison, root density of root hairs in one region of the soil than nodules develop on the same plant when induced by would otherwise be achieved. The importance of rhizobia, and contain (as in other legumes) peripheral contact surface maximization in the uptake of limiting rather than central vascular tissues (Squartini et al. nutrients is also a main issue for structures such as the 1993). From both anatomical and aetiological stand- cluster roots (Lambers et al. 2006; Lambers et al. points, shovels also appear rather distinct from 2008). Other structure-function studies also indicate proteoid roots (Purnell 1960; Gardner et al. 1982), that clustered surfaces are better designed for the referred to also as cluster roots (Lambers et al. 2003; concentration of root exudates than they are for the Lamont 2003; Skene 2003; Shane and Lambers uptake of nutrients (Gardner et al. 1982) (4) Shovels 2005), which are stacked clusters of rootlets formed concentrate Ca++intracellularly, and eventually these in different plant species in response to P or Fe calcium mineral deposits grow into crystals that are starvation. However, both shovels and cluster roots unlikely to be re-used for further physiological appear to share physiological clues in the context of functions, as the mature shovels dry and detach from solubilization of limiting nutrients (2) The shovels the root. Moreover, the Ca content of H. coronarium eventually develop an inward-curved “shovel-like” stems and leaves is not different from that occurring shape that is due to an asymmetrical distribution of in other legumes such as clover or alfalfa when grown cell volume of the cortical cells, being larger on the in the same soil (Bolli 1951). Therefore, the precip- convex side. (3) The high density of root hairs on the itated calcium storage of the root shovels behaves as a concave side suggests efficient absorbing functions, dead-end sink that removes calcium from the sur- and the larger cortical cells on the convex side are rounding soil. Infrared spectra, STEM X-ray micro- consistent with a storage parenchyma histology. In analysis and crystals solubility behaviour all indicate order to understand the enhanced functions acquired that the anion of the calcium salt that precipitates by the transition to a concave shape, an analogy with within shovels varies according to its local abundance a familiar object such as a cushion pin hairbrush, is in the environment. The phenomenon of calcium helpful, notice how when pressing the tips until accumulation by plant roots is reminiscent of litera- inverting the surface from convex to concave, the ture reports on the occurrence of calcified roots as a pin tips would become closer and denser towards a peculiar geopedological feature which is observed in central point. The same applies for root hairs in the rendzinas, calcic xerosols and cambisols (Jaillard Plant Soil (2009) 322:25–37 35 et al. 1991). Calcified roots, preserved in layers of account the role of iron oxide as an inducer of their different age, are indicated as remnants of plant formation. Hedysarum coronarium grows remarkably species able to accumulate calcium carbonate in well in alkaline soils, always showing a deep green limestone-rich soils. A rhizogenic contribution to the foliage without chlorotic symptoms. However, iron soil geochemistry has also been defined (Jaillard availability as a plant nutrient is dramatically limited 1984; Hinsinger et al. 2003). by its insolubility at alkaline pH values, leading to In their native calcareous setting, sulla shovels iron oxides or hydroxides precipitation. Unlike other accumulate calcium carbonate deposits, whereas they legumes, we have shown that sulla can efficiently accumulate calcium phosphate deposits when grown in acidify alkaline root environments to near neutrality. the lab using Fahräeus solution, that contains phos- But, in calcareous soils (a typical habitat where H. phate rather than carbonate anions. Therefore, the coronarium thrives and forms many shovels), other nature of the crystalline end product does not seem to plants face a main hindrance while attempting to be genetically defined, but rather the mechanism of lower the rhizospheric pH, because abundant calcium crystal formation involves the scavenging of the is a key element of the soil’s pH buffering capacity, abundant external source of calcium into a precipitat- well known as the carbonate buffer and shown by the ing salt using the most available anion. We suggest that following reactions: the main function of these organs, no matter which salt CO þ H O ! H CO is formed, is basically to remove calcium from the 2 2 2 3 CaCO þ H CO ! Caþþ þ 2HCO: vicinity of the root and store it as inert disposable 3 2 3 3 structures. (5) In-vivo tests showed that sulla is An increase in CO2 content in the soil atmosphere particularly well suited to neutralize the pH of both or a decrease in soil solution pH will drive the acidic and alkaline root environments in contrast to reactions to the right; as a consequence, carbonate ++ other legumes like white clover which exhibited little will dissolve and move as Ca and HCO3 with the capability to counteract the basic reaction. In relation to soil water. Precipitation of carbonate occurs under the goal of nutrient solubilization, and in line with conditions that drive the reaction to the left, i.e. a strategies also displayed by plants capable of forming lowering of pCO2, a rise in pH, and an increase in ion cluster roots (Lambers et al. 2006) it can be envisaged concentration to the point where saturation is reached that Hedysarum coronarium could release carboxy- and precipitation occurs. lates or other rhizosphere acidifying organic mole- In this scenario, the protons extruded by plant roots cules. Some other legumes such as chickpeas and may not be effective in acidifying since, so long as lupin have been reported to express such physiolog- calcium is abundant and soluble, the equilibrium ical strategies (Veneklaas et al. 2003). The importance between the above forms will drive hydrogen ions of root-mediated pH changes in the rhizosphere and into H2CO3. But once calcium is locally removed and their impact on calcareous soils in relation to nutrient stored internally (as occurs with shovel crystals), the uptake has been reviewed (Hinsinger et al. 2003). removal of this pivotal element of the system’s Moreover, from lessons taught from the examples of buffering capacity will allow acidification to be cluster roots, a dense mass of root hairs appears to be effective. Therefore, in the soil surrounding the an essential asset to change the pH of the surrounding shovels, the pH will drop to levels at which iron can soil (Lambers et al. 2006) and the shovel inward- regain its solubility and be readily adsorbed by the folded shape appears to follow this model. (6) Shovel extensive root hair mane present on the shovel’s formation is developmentally induced by direct concave side. contact with water-insoluble iron oxides. Soils can Therefore, it appears meaningful that iron oxide be contain abundant iron oxides, as shown by their the triggering signal for shovel formation because its frequent reddish brown colours. Indeed, while iron in presence in the ferric state signals to the plant that: (1) soils can easily amount to 38,000 ppm, in its different iron is present and its uptake can therefore be forms the corresponding iron in solution can be as fruitfully attempted; (2) the pH is alkaline having low as 0.001 ppm (Lucena 2003). led to iron precipitation. Therefore, removing calcium The results described here led us to postulate the will enable the plant to solubilize iron locally. In this following model for shovel function, which takes into respect, sensing oxidized iron would be interpreted by 36 Plant Soil (2009) 322:25–37 the plant as the most direct and univocal indication of operate with variable degrees of efficiency (Lioi and being in an alkaline soil where shovels are needed to Giovannetti 1987). enable rhizospheric acidification leading to an effec- This model illuminates the mechanisms of adapta- tive iron uptake. The mere basic pH would not be an tion of H. coronarium, contributes to an understand- equally efficient signal as it would not warrant iron ing of its physiological success in stressed, marginal uptake once alkalinity is counteracted. areas where other vegetation cannot colonize, and Alkaline soils include different subsets ranging points to a useful source of genes and physiological from calcareous (CaCO3 content higher than 10 g/kg strategies for engineering other plants to tolerate and a pH typically in the 8–8.5 range) to sodic multiple chemical stresses and maintain sustainable (exchangeable sodium percentage higher than 15% of productivity. The traits displayed by a plant like sulla the cation exchange capacity, and pH above 8.5) and are in this respect particularly relevant for an saline-sodic (having the same exchangeable sodium agriculture increasingly challenged by global warm- percentage, an electrical conductivity of the soil water ing and land desertification. extract higher than 4 mS/cm, and a pH below 8.5). In order to verify the model prediction we surveyed the Acknowledgements The authors are grateful to Piergiorgio Vergamini for assistance in the IR spectroscopy. Serenella data available on the sites where the plant occurs and Nardi is acknowledged for helpful discussions. A preliminary verified in which of these sites shovel formation had account of this report was presented at the XI International mostly been recorded. One of the prime habitats of Symposium on Iron Nutrition and Interactions in Plants, held in sulla is in South-Western Tuscany. In those pliocenic Udine, Italy on June 23rd-28th 2002. clays shovels are always particularly abundant and developed. Moreover, those are the sites where these References peculiar structures had historically been signalled for the first time and where their occurrence is even part Arnott JH, Pautard FGE (1970) Calcification in plants. In: Schraer of the farmers’ popular knowledge. Detailed soil H (ed) Biological calcification: cellular and molecular analyses of this region (Carloni et al. 1973) allow to aspects. Appleton-Century-Crofts, New York, pp 375–446 pinpoint the features of the typical shovel-forming Ballatore GP (1963) La coltivazione della sulla. Informatore Agrario 52:5–15 horizon. By focusing on the layer at a depth between Beringer JE (1974) R factor transfer in Rhizobium legumino- 0 and 30 cm, where these structures form, the usual sarum. 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