Ecophysiological properties of a cluster root- forming plant, Helicia cochinchinensis (Proteaceae) grown on Miyajima Island, Japan


 Aims The family Proteaceae is one of the dominant families in nutrient-impoverished habitats in the Southern hemisphere, and less common in the Northern hemisphere. Helicia cochinchinensis Lour. is the only Proteaceae species in Japan. This study aimed to unveil the ecophysiological properties of H. cochinchinensis grown on Miyajima Island, Hiroshima, Japan.Methods Phosphorus (P) status and dynamics of soils in H. cochinchinensis habitats were measured. Plant P and nitrogen (N) concentrations of leaves were measured after digestion. Roots and rhizosheath soil were collected to assess root morphology and root exudates.Results Available P (Olsen-P) in soils in habitats of H. cochinchinensis was 0.46–3.7 mg P kg-1 soil. Citrate was the major carboxylate in root exudates and its concentration increased during cluster-root formation. Acid phosphatase activity was greater at the surface of cluster roots that on the surface of other roots and bulk soil, especially for mature cluster roots. Sparingly soluble organic P concentrations decreased in the rhizosheath soil of mature cluster roots. The P concentrations of H. cochinchinensis leaves were relatively low; 0.34–0.69 mg P g-1 DW and 0.15–0.29 mg P g-1 DW in mature and senesced leaves, respectively. The P demand of H. cochinchinensis was less than that of nearby trees, showing greater P-remobilization efficiency.Conclusions Phosphorus mobilization from unavailable P by cluster roots supported P uptake by H. cochinchinensis, and P remobilization from senescing leaves contributed to sustain growth under P-deficient conditions.


Introduction
The Proteaceae family comprises woody dicots that are distributed mainly in Australia and South Africa, which were part of Gondwana (Lambers et al. 2012a). Southwestern Australian native Proteaceae such as species in the genera Banksia, Grevillea, and Hakea are adapted to extremely nutrient-impoverishment soils that are especially low in phosphorus (P) (Lambers et al. 2011). The formation of a bottle-brush like root architecture, so-called 'cluster roots', is a strategy to adapt to P-de ciency (Neumann and Martinoia 2002). Cluster roots consist of very dense rootlets that are several millimeters long, produce a high density of root hairs on every rootlet, and release carboxylates in an exudative burst (Shane and Lambers 2005). Consequently, cluster roots can mobilize and take up P from the rhizosphere. Both morphological and physiological alterations in the cluster roots are important for P uptake. Exudative bursts such as of carboxylates and phosphatases are strategies to mobilize unavailable P, such as sparingly soluble inorganic P and organic P, in soils (Neumann and Martinoia 2002;Wasaki et al. 2003).
Phosphorus remobilization from senescing leaves is a well-known strategy for growth under P-de cient conditions. Some Proteaceae plants remobilize P from senescing leaves highly e ciently (Denton et al. 2007). Although global average values of the P-remobilizing e ciency (PRE) are about 50% (Lambers and Oliveira 2019), Banksia and Hakea species show a much greater PRE: greater than 80% (Denton et al. 2007;de Campos et al. 2013).
Replacement of phospholipids with galactolipids and sulfolipids during leaf development has been found in three Banksia and three Hakea species as an e cient P utilization strategy (Lambers et al. 2012b). Furthermore, P re-use from rRNA in same leaves during development has been shown in three Banksia and three Hakea species (Sulpice et al. 2014). The authors suggested that these properties contribute to the extremely high photosynthetic phosphorus-use e ciency of Proteaceae species.
A Southern South American Proteaceae in the genus Embothrium, naturally growing in young soils with high content of total P but low P availability, shows higher leaf P concentration and lower Premobilization e ciency than southwestern Australian Proteaceae species (Hayes et al. 2021). It has been suggested that cluster roots of Embothrium play a major role in the uptake of nitrogen (N) rather than P (Fajardo and Piper 2015;Piper et al. 2013 This study aimed to unveil the ecophysiological properties involved in soil P mobilization by their roots and the P-remobilization e ciency of Helicia cochinchinensis growing in a natural habitat on Miyajima Island, Japan.

Study Sites and Sampling
The sampling site of Helicia cochinchinensis was Miyajima Island (Fig. 1), which has a warm and wet climate (34°17'N, 132°17'E, 20 m above sea level, annual precipitation 1,644 mm, annual average temperature 15.8˚C). Two planted sites in the Miyajima Natural Botanical Garden (sites A and B), which is near natural habitats on the island, and three natural habitats on Miyajima Island (sites C, D, and E) were selected as study sites. Bulk soils near H. cochinchinensis plants at sites A-E were sampled and air-dried for analyses. The rhizosheath soil of H. cochinchinensis was sampled: cluster roots were collected from mature trees and gently shaken to remove non-rhizosheath soils adhered to the roots. The roots containing rhizosheath soil were transferred to 50 mL polypropylene tubes and shaken thoroughly. The roots were then removed from tubes and washed well with deionized water and used immediately for physiological analyses. Some roots were stored for DNA barcoding at -20°C until analysis. The rhizosheath soil was also air-dried.
Mature leaves of H. cochinchinensis and nearby trees were collected for elemental analysis. Senescing and dead leaves were also sampled from parts of trees. Fully expanded, yellowing, defoliated leaves were de ned, respectively, as mature, senescing, and dead leaves. Leaves used for element analyses were oven-dried for three days at 70°C.

Soil Analysis
All soil samples were sieved with a 2-mm sieve to remove large particles and roots. The samples were later used for analysis. Soil pH was determined in a water suspension (soil water ratio of 1:2.5) after shaking for one hour.
Total P of soils was determined using the vanadomolybdate blue method (Murphy and Riley 1962) after digestion of the sample with H 2 SO 4 -H 2 O 2 . Available P (Olsen P) in the soil was analyzed according to Olsen et al. (1954). Phosphorus fractionation was conducted according to Hedley et al. (1982) with one modi cation in that a process to analyze soluble P was omitted, because Olsen-P already indicated a similar fraction.
Sand Culture of H. cochinchinensis Helicia cochinchinensis seedlings were collected on Miyajima Island and transferred to a cylindrical pot (190 mm in height and 160 mm in diameter) containing washed river sand. They were cultivated in a greenhouse through one year with continuous supply of a nutrient solution without P every seven days.
Well-washed plants were used as -P individuals for observation of root morphology, collection of root exudates, and activity staining of acid phosphatase. After collection of root exudates, all plants were transferred to nutrient solution containing P (320 µM) and cultured in a greenhouse. After six days of P supply, all plants were used as +P individuals for collection of root exudates and activity measurements of acid phosphatase.

Analysis of Cluster Roots
For the measurements of carboxylate exudation, exudates of whole roots of sand-cultured plants were collected by soaking in 200 mL of distilled water for 3 h. Citrate and malate concentrations in root exudates were analyzed using an enzymatic method (F-kit, Roche, Basel, Switzerland). Numbers of cluster roots were counted for all analyzed plants.
Secreted and root-surface acid phosphatase activities of cluster roots were analyzed as below. The fresh roots were excised and soaked in distilled water for 1 h to remove leakage from root sections. The roots were transferred to new tubes containing 50 mL of distilled water for 3 h to collect root exudates. An aliquot (0.5 mL) of root exudates was added to 1.5 mL of 0.2 M acetate-NaOH buffer (pH 5.5), and 0.5 mL of substrate solution (10 mM p-nitrophenyl phosphate) was subsequently added. After incubation at 30˚C for 30 min, the reaction was stopped by adding 2.5 mL of 0.2 M NaOH. To analyze total activity of acid phosphatase, the roots were immersed in a solution contained 30 mL of acetate-NaOH buffer, 10 mL of distilled water, and 10 mL of substrate solution at 30˚C for 30 min. The reaction was stopped after removal of the root system from the solution by adding 50 mL of 0.2 M NaOH. Acid phosphatase activities were measured by spectrophotometry at 420 nm as increase of the p-nitrophenol concentration. Acid phosphatase activities on the root-surface were calculated by subtraction of secreted phosphatase activity from total activity. Activity staining of phosphatase was conducted using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a substrate. Washed cluster roots were soaked for 30-180 min in a staining solution containing 50 mM sodium acetate (pH 5.3), 10 mM MgCl 2 , and 0.1 mg mL −1 BCIP. Blue-stained samples were observed using a stereoscopic microscope.

P Fractionation of the Soil
To collect cluster roots exhibiting rapid exudation ability, the decrease of pH was used as the index. The pH alteration by root exudates was visualized using a pH indicator according to Shane et al. (2006).
Brie y, roots collected in the eld were directly placed on a 0.75% (w/w) agar gel containing 0.008% (w/v) of bromocresol purple as pH indicator.
For determination of soil P pools, sequential fractionation was conducted using the method described by Hedley et al. (1982) with minor modi cation. Brie y, 0.5 g of soil was weighed and extracted sequentially by shaking for 16 h with solutions as below. First, 30 mL of 0.5 M NaHCO 3 was used after adjustment to pH 8.5. A second and third extraction used 30 mL 0.1 M NaOH (NaOH-P) and 20 mL 1 M HCl, respectively. Inorganic P (Pi) extracted by NaHCO 3 and NaOH was measured as NaHCO 3 -Pi and NaOH-Pi, respectively. Total P extracted by NaHCO 3 , NaOH and HCl was measured after digestion by autoclaving in 1.8 M H 2 SO 4 with (NH 4 ) 2 S 2 O 8 . Organic P (Po) in these fractions was calculated by subtraction the Pi from total P. Total P was determined by the vanadomolybdate blue method, after digestion.

Elemental Analyses
Dried leaves were ground using a mortar and pestle. A sample of approximately 50 mg was digested using H 2 SO 4 -H 2 O 2 . Digested samples were used for analysis of total P and N concentrations, respectively, using the molybdate blue method and the indophenol method (Bolleter et al. 1961). The P-remobilization e ciency (PRE) was calculated as the difference between mature and senesced leaf P concentrations divided by the mature leaf P concentration (Killingbeck 1996).

Statistical Analyses
All cultivations were replicated more than three times. Student's t-test and Tukey's test were used with SPSS version 16J (SPSS Inc., Chicago, IL, USA). Signi cance was accepted at P<0.01 or P<0.05.

Results
Helicia cochinchinensis habitat and the soil P status Mature H. cochinchinensis plants were about 20 m in height in Miyajima ( Fig. 2A), owering in early August (Fig. 2B). The shape of the seeds is elliptical with 1 cm in length of the longest dimension (Fig.   2C). The morphology of typical 'simple type' cluster roots (Fig. 2D, E, F) was found like in the genus Hakea (Shane and Lambers 2005). Cluster roots of H. cochinchinensis were found mainly within 10 cm from the soil surface. All individuals formed cluster roots, regardless of the differences of cultivation. Their cluster roots tightly bound soil particles and organic matter. The widths and lengths of clusters reached more than 1 cm and 3 cm, respectively (Fig. 2E, F). The cluster roots were able to nd from naturally grown H. cochinchinensis in all seasons.
We measured total and Olsen P concentrations of bulk soil sampled at ve sites in H. cochinchinensis habitats ( Table 1). Total and Olsen P in soils in H. cochinchinensis habitats were, respectively, 135-329 and 0.46-3.7 mg P kg −1 soil. Both values were highest at H. cochinchinensis-planted site A, although these were less than one-tenth of the values in cropland close to Miyajima Island. The total P concentration in Miyajima was several times higher than the value in southwestern Australia, where Proteaceae plants are predominant. However, the level of Olsen-P in Miyajima was equivalent to or slightly higher than that in southwestern Australia, suggesting that Miyajima is extremely poor in available P. Soil pH was 5.3 or similar at all analyzed sites.

Properties of H. cochinchinensis cluster roots
Citrate and malate exudation rates were analyzed using sand-cultured H. cochinchinensis plants ( Table  2). Both citrate and malate exudation in -P plants were signi cantly greater than those in +P plants, suggesting that these carboxylate exudations were induced by P-starved conditions in H. cochinchinensis. The number of cluster roots per plant was positively correlated with both citrate and malate exudation rates (Fig. 4), indicating the contribution of cluster roots to carboxylate exudation. Acid phosphatase activities were measured in different compartments. The results show that all activities were signi cantly higher in -P plants ( Table 2). Interestingly, the activity of root-surface phosphatase was higher than that of root-secreted phosphatase. Activity staining of phosphatase was conducted using BCIP as a substrate. Activities were detected at the mature cluster root stage (Fig. 4AB). High intensity of blue-stained phosphatase was also found in the zone with high-density root hairs (Fig. 4C). The pH change around cluster roots of H. cochinchinensis grown in soil was also visualized (Fig. 4D). The pH reduction was stronger in the rhizosheath of mature cluster roots than that of senesced cluster roots. To investigate the effects of root exudates from cluster roots on the P dynamics of actual rhizosheath soils, sequential fractionation analysis of P was conducted for the bulk soil and the rhizosheath soils of cluster roots, which showed strong pH reductions. Total P concentration of the rhizosheath soil was mostly half of that of bulk soil, and concentrations of all fractions were lower in the rhizosheath soils (Fig. 5). Surprisingly, a reduction of the NaOH-Po fraction in rhizosheath soil was common. This suggests that insoluble organic P was the major form of P mobilized by the cluster roots.  The P concentrations of mature leaves of native trees were compared at the family level (Fig. 6) was the highest value and 0.45 mg P g −1 DW was the median. This value was fourth from the lowest among analyzed plants.
The P and N concentrations of mature, senesced, and dead leaves were compared among H. cochinchinensis, Camellia japonica, and Michelia compressa which showed higher PRE. Both concentrations were low in senesced and dead leaves and high in mature leaves in all plant species (Fig.  7). A linear correlation was found between N and P concentrations in all plants. The slopes for Camellia and Michelia were similar. Helicia cochinchinensis showed a lower slope than other species did (Fig. 7), suggesting that the relative demand of P to N in H. cochinchinensis was less than that in other species, even in those planted at the same places.

Discussion
Phosphorus mobilization in the rhizosheath of cluster roots Helicia cochinchinensis formed simple cluster roots (Fig. 2), as do most Proteaceae (Shane and Lambers 2005). This morphology has bene ts for P uptake via increasing of root surface areas and massive release of root exudates. Physiological alteration in the cluster roots of H. cochinchinensis was similar to that of other Proteaceae from Australia, South Africa and South America (Shane and Lambers 2005).
Cluster roots caused a strong decrease of pH in the rhizosheath (Figs. 4D, 5) and showed massive carboxylate exudation (Fig. 3). Earlier reports have described that the pH decrease and carboxylate exudation contribute to P mobilization in the rhizosheath ( Phosphatases cleave phosphate ester bonds of a broad range of organic P compounds in soils, and can contribute to the supply of inorganic P. Both secreted and root surface-bound phosphatases mobilize organic P in the rhizosheath (Wasaki et al. 2003). In H. cochinchinensis, the phosphatase activities on the root surface were predominant rather than the root secreted phosphatases ( Table 2). This is inconsistent with previous studies on hydroponically-cultured crop plants, in which secreted phosphatases showed higher activities than cell wall-bound phosphatases (e.g., Tadano and Sakai 1991). The reason may be the characteristics of the cluster roots. The cluster roots develop dense root hairs on their rootlets to increase the root surface area. Thus the rhizosheath of cluster roots has a larger volume than other roots of the same weight. The stronger activities on root surfaces seems reasonable for cluster root-forming plants, such as Proteaceae. Indeed, mature cluster roots and their root hair cells showed stronger activities of phosphatase by the activity staining of root-surface phosphatases (Fig. 4). The phosphatase activity of the entire root system of Embothrium coccineum was less than 150 µmol g −1 DW h −1 (Delgado et al. 2013), suggesting that H. cochinchinensis had markedly high activities (Table 2). Given the higher organic P content in the soil of H. cochinchinensis habitats in Miyajima (Fig. 5), it seems reasonable for the role to mobilize the organic P in the rhizosheath soils.
Effects of P-mobilizing abilities in the rhizosheath of H. cochinchinensis on actual soils were estimated by a sequential fractionation. Surprisingly, more than half of total P in bulk soil disappeared in the rhizosheath soils (Fig. 5), suggesting that P-mobilization abilities of the rhizosheath signi cantly contributed to P acquisition from 'unavailable' P in soil. NaOH-Po, which was a drastically decreased fraction in the rhizosheath soils, contained phytate-P, the most abundant organic P in soils ( Phosphorus remobilization from senescing leaves The critical level of P in crop plants to avoid P de ciency is 2 mg P g −1 DW (Marschner 2012, Tadano and Sakai 1991). The P concentration of mature leaves of native trees in Miyajima was much lower (Fig. 6, Table 3). The median of P concentration of H. cochinchinensis was fourth from the lowest among the plants investigated on Miyajima Island (Fig. 6), and mostly average for Proteaceae (Westoby and Falster 2021). This suggests that many plant species in Miyajima Island including H. cochinchinensis can tolerate low-P conditions and function at low leaf P concentrations.
Phosphorus remobilization of Proteaceae in southwestern Australia is extremely important for growth in soil containing very little P (Lambers et al. 2012b(Lambers et al. , 2015. The P-remobilization e ciency of H. cochinchinensis from senesced leaves was greater than 50% for all investigated trees (Table 3). Phosphorus remobilization of H. cochinchinensis was greater than that of nearby trees of other families, although the PRE was lower than that of southwestern Australian species.
The Southern South American Proteaceae Embothrium coccineum naturally occurs in volcanic soils, which have a high total P concentration but low P availability due to strong P sorption. These plants show only 42% P-remobilization e ciency, although this value is higher than that of nearby trees (Fajardo and Piper 2015). The P-remobilization e ciency of H. cochinchinensis was markedly higher than that of Embothrium coccineum (de Campos et al. 2013). Higher PRE in Proteaceae than in other families commonly occurs in three different regions (Australia, Chile, and Japan). Mature leaves of wide range of Proteaceae showed low P concentration (Hayes et al. 2021). These facts suggest that e cient P use is important for P nutrition in Proteaceae. P-N interactions suggest a relatively low P demand in H. cochinchinensis. The slope of the correlation for H. cochinchinensis tended to be lower than those of the other two trees (Fig. 7), suggesting that P demand of H. cochinchinensis was less than that of nearby trees showing high P use e ciency. Wright et al. (2004) reported that mature leaf N:P ratios of southwestern Australian Proteaceae was 24.4, whereas those of tested species on Miyajima Island were lower. Nitrogen concentrations of senesced leaves were lower than those of mature leaves (Fig. 7). Mature leaf N concentrations were higher among Proteaceae (Westby and Falster 2021). Resorption of N from litter to green leaves was low in southwestern Australian Proteaceae plants (Lambers et al. 2012a). This suggests that both P and N are limiting nutrients for Miyajima trees, although P is the strongest limiting nutrient, as is the case for southwestern Australian species.

Concluding Remarks
Morphological and physiological properties of cluster roots of H. cochinchinensis on Miyajima Island support P uptake from poorly available P sources in the soil like those of other Proteaceae. Actually, P remobilization from senescing leaves and less P demand in leaves may sustain the growth of H. cochinchinensis under P-de cient conditions. The ecological and physiological importance of H.   Relationships between cluster-root formation and carboxylate exudation. A, citrate exudation; B, malate exudation.

Figure 5
Phosphorus fractionation and pH of rhizosheath soils of cluster roots and bulk soils.
Page 19/20 Figure 6 Phosphorus concentration of mature leaves of native trees on Miyajima Island.