Root Morphology and Anatomy of Field-Grown Erianthus arundinaceus

Erianthus species are perennial C4 grasses with such high biomass productivity and high tolerance to environmental stresses that they can be grown in marginal land to supply raw material for cellulosic bioethanol. Because high biomass production and strong tolerance to environmental stresses might be based on their large and deep-root system, we closely examined the morphology and anatomy of roots in first-year seedlings of field-grown Erianthus arundinaceus. The deep-root system of E. arundinaceus consists of many nodal roots growing with steep growth angles. Diameter of nodal roots with large variations (0.5 5 mm) correlates with the size and number of large xylem vessels. The microscopic observation shows that the nodal roots with dense root hairs developed soil sheath, hypodermis with lignified sclerenchyma in the outer cortex, and aerenchyma in the mid-cortex. In addition, starch grains were densely accumulated in the stele of nodal roots in winter. In the first year, E. arundinaceus developed less lateral roots than other reported grass species. The lateral roots formed a large xylem vessel in the center of the stele and no hypodermis in the outer cortex. Morphology and anatomy of E. arundinaceus root were discussed with reference to strong tolerance to environmental stresses.


Introduction
Energy consumption in the world based mainly on fossil energy has doubled in the 20th century, which has raised the concentration of carbon dioxide in the atmosphere.Reducing dependence on petroleum is, therefore, essential for combating global warming [1]- [3].Bioethanol produced from plant biomass is a renewable and alternative fuel that can be a countermeasure to global warming when produced in appropriate ways [4] [5].Rapid increase of bioethanol production from cereal crops in the world [6], on the other hand, may cause possible competition for cereal crops between food and bioethanol [7] [8].Non-edible cellulosic energy crops (e.g.switchgrass, napier grass, Erianthus spp.and Miscanthus spp.), therefore, are expected to be grown on degraded lands as raw material crops for bioethanol instead of edible energy crops (e.g.sugar cane, maize and cassava) [3] [9]- [14].
Erianthus species which have been used as genetic resources for breeding sugarcane [15]- [18] and as potential forage crops [16] [19] might be potential raw crops for bioethanol [11] [12] [20], because they have huge biomass as well as high tolerance to biotic and abiotic stresses, such as drought, waterlogging, and soil hardness.Although morphological and anatomical characteristics of roots have possible relation to tolerance of the plant to such environmental stresses [21]- [31], there has been quite limited information on their roots.In this study, morphology and anatomy of roots in a promising strain, KO-1 of E. arundinaceus originated from Tanegashima Island (30˚N, 130˚E) in a subtropical area of Japan to discuss their possible contribution to the high productivity and strong tolerance to soil-related stresses.

Material and Methods
Five-month-old seedlings of E. arundinaceus (strain KO-1; [32]) grown in a greenhouse at the National Agricultural Research Center for Kyushu Okinawa Region (Kumamoto, Japan) were transplanted at planting density of four plants per m 2 (0.5 m × 0.5 m spacing) in a field of the Institute for Sustainable Agro-ecosystem Services, The University of Tokyo (35˚43'N, 139˚32'E; Nishitokyo, Japan) on July 29, 2009.Before planting the field of humic Andosol was fertilized at rate of N: 100, P 2 O 5 : 150, K 2 O: 130 kg•ha −1 before planting.
Plant height and stem number of 12 selected plants were recorded every two weeks.Roots were taken by monolith method [33] [34] at 80 and 141 days after planting (DAP).In addition, five soil cores (5 cm in diameter × 0.6 m deep) were collected from locations adjacent to Erianthus plants and in interrows using the DIK-110B liner sampler (Daiki Rika Kogyo Co., Ltd.).Soil monoliths and soil cores were divided by 15 or 30 cm increments to wash roots out carefully.The number of nodal roots from the plants collected by the monolith method was counted.Images of roots were scanned at 200 dpi using the image scanner (Epson Expression 10000XL) to measure root length including laterals as a function of root diameter using WinRHIZO Regular (Regent Instruments Inc.).Thereafter the roots were dried at 80˚C for three days to weigh, and specific root length (ratio of total root length to total dry weight) was calculated.
In addition, Erianthus plants with roots were taken out using a shovel at 148 DAP (December 24, 2009).Diameters of randomly selected nodal roots of a dozen were measured using a digital caliper at ca. 5 cm from their base.Twenty-one additional nodal roots were randomly chosen for anatomical observation.Freehand cross sections of these fresh roots were observed using a light microscope (BX-60, Olympus Corp.) to record the diameters of root, stele and large xylem vessels, the pole numbers (i.e., number of protoxylem) and large xylem vessels.Equivalent diameter of large xylem vessels was calculated as the biquadratic root of sum of the fourth power of the large xylem vessel [Σ(diameter of large xylem vessels) 4 ] 1/4 on the basis of the Hagen-Poiseuille equation [35].Some of the cross sections of nodal roots were stained with phloroglucinol-HCl [36] and potassium iodide solutions [37] to detect lignin and starch, respectively.Furthermore, cross sections of several lateral roots were observed microscopically to measure thickness and stele diameter.Erianthus plants with roots were again harvested in summer using a shovel (349 DAP; July 13, 2010) to observe starch accumulation in nodal roots.

Plant Growth
Plant height and stem number increased until autumn of 2009 (85 DAP) and the plant leveled off in winter.Shoot dry weight, however, increased by 1.7-fold (80 DAP -141 DAP) during winter (Table 1).The number of nodal roots was almost constant throughout the growing period (ca.70, Table 2).Most nodal roots emerged from relatively thick stems to grow downwards (Figure 1(a)), while many small tillers lacked nodal ones.

Root Diameter and Branching Index
Diameter of nodal roots at 148 DAP widely varied (Figure 1(b)), ranging from 0.5 to 5 mm (from 1 to 3 mm even among young nodal roots less than 10 cm). Figure 2 shows the distribution of root length as a function of their diameter at 80 DAP (including lateral roots).Considering the root diameter from both WinRHIZO measurement (see above) and microscopic observation (see the next section), we recognized roots of less than 0.5 mm diameter as lateral ones.The branching index (i.e., [total length of lateral roots]/[total length of nodal root axes]; [38]) was 2.8 and 1.9 in 0 -15 cm and 15 -30 cm soil layer, respectively.The branching indices of roots collected by the core sampling method at 0 -15 cm, 15 -30 cm, and 30 -60 cm soil layers were 5.6, 2.4, and 3.3, respectively.The specific root length (i.e., root length per unit root weight) was 7.4 m•g −1 at 80 DAP decreasing to 4.4 m•g −1 at 141 DAP (Table 2).

Anatomy of Nodal Roots
Nodal roots were tightly covered with the soil sheath [28] [39] adhering to the root epidermis (Figure 3 Hypodermal cells in the cell layer below epidermis were similar in size to epidermal ones, whereas the second layer of the hypodermis consisted of smaller sclerenchyma cells (Figure 3(d)).The sclerenchyma with lignified cell walls was partly double layered or multiseriate (Figure 3(d)).Most nodal roots formed well-developed aerenchyma in the mid-cortex (Figure 3(b), Figure 3(e)).Endodermal cells had thick inner tangential cell walls heavily lignified, and sometimes showed periclinal division (Figure 3(f)).
The stele (central cylinder) consisted of three parts: a marginal cylinder of multiseriate lignified pericycle, a ring of conducting tissues, and parenchymatous central pith (Figure 3(b)).Pericycle is not easily idendified and it is composed of 2 -4 layers of oval cells with thick and heavily-lignified walls, which is different from other cereal crops (Figure 3(f)).Xylem and phloem poles were located in a circle at regular intervals in the outer parts of the stele, and large xylem vessels were inside (Figure 3(b), Figure 3(f)).The small and large xylem could be protoxylem and metaxylem, respectively, according to their size and position.The center of the stele is occupied by unlignified parenchymatous cells with starch grains stained with potassium iodide (Figure 3(g)) at 148 DAP (December, 2009).Some roots had such starch grains even in oval cells in the lignified marginal cylinder of multiseriate pericycle.Almost no starch grains, in contrast, were observed in the stele of Erianthus roots harvested in summer (349 DAP), except in a few aged roots (Figure 3(h)).
The relationships between root diameter, stele size, and number of xylem poles were analyzed for the 21 nodal roots.Stele diameter was closely correlated to root diameter (Table 3, Figure 4(a)).Moreover, the number of xylem poles and large xylem vessels (Figure 4(b)) and mean diameter of large xylem vessels (Figure 4(c)) were positively correlated with root diameter (Table 3).The equivalent diameter of the xylem vessels, therefore, could be estimated from the root diameter (Figure 4(d)).

Anatomy of Lateral Roots
Lateral roots have neither lignified hypodermis or aerenchyma as in the cortex of nodal roots, while the endodermis with thickened inner tangential cell walls was developed (Figure 3(i)).There was one large xylem vessel in the center of the stele where no starch grain was accumulated.The ratio of stele diameter to root diameter in The values indicate means and standard errors of four monoliths.Root density and specific root length were calculated using the mean values of root length and dry weight.

Discussion
Preliminary observation on three-year-old Erianthus (same strain KO-1) showed that their root system has at least 2.6 m depth and nearly 20 % of roots distributed deeper than 1.5 m [40].Root distribution in the fibrous root system of grasses is determined by a combination of factors such as root growth direction [41] [42], root length [43] [44], and root penetrating ability into hard soil [45] [46].It has already known that thick nodal roots of cereal crops, in general, tend to elongate vertically [41] [42] [47].In the present study, the first-year plants of E. arundinaceus possessed many thick roots with diameter over 2 mm even at the early growth stage (Figure 2, Figure 4), and most nodal roots mainly elongated vertically (Figure 1(a)).Plant species and genotype with such thick nodal roots also have advantage to penetrate into deeper soil layer [48] [49].Matsuo et al. [16] suggested that the roots of Thailand strain of Erianthus have high ability to penetrate hardpan based on their field study in Northeast Thailand.Such morphological traits of Erianthus roots could lead to develop deep root system, which has advantage to environmental stresses for example drought by extracting soil water remaining in deep soil layer.Nodal roots of Erianthus was characterized by small specific root length in the present study, which suggests larger diameter and less branching of nodal roots in the first year (Table 2, Figure 2).Thick nodal roots of E. arundinaceus have numerous large xylem vessels with linified cell walls which are adaptive to support huge aboveground part as reported in maize [50].Less branching was confirmed by lower branching index (1.9-5.6) compared with cereal crops such as wheat (3 -10 in nodal roots about 145 days after sowing; [51]), or maize (10 -150 in mature nodal roots ca.120 days after sowing; [52]).High specific root length and low branching index of E. arundinaceus roots suggests advantage in nodal root development rather than lateral root formation at early growth stages in Erianthus (Table 2).Our preliminary observation on three-year-old Erianthus, however, showed the development of shallow nodal roots with numerous lateral roots (data not shown) as well as deep nodal roots.Root system development could also change with growth stages in other cereal crops [51] [53] [54].Root system development in younger Erianthus could be characterized by thick and deep nodal roots with less branching as the "buttress roots" in sugarcane; and older Erianthus may develop "superficial roots".
Equivalent xylem vessel diameter defined as a function of the fourth power of mainly large xylem vessel diameter (see Material and Methods) is thought as an index of total conductivity in roots having xylem vessel with different number and size [35] [55].The amount of vascular tissues often correlates to the root or stele diameter [55]- [58] as was confirmed in Erianthus roots in this study (Table 3, Figure 4(b), Figure 4(c)).Equivalent xylem vessel diameter has linear relationship to root or stele diameter (Figure 4(d)), which suggests Erianthus root system could have higher water conductivity based on thicker and larger nodal roots comparing with other cereal crops.Vessel anatomy also suggests that Erianthus root system has high potential in absorption and transportation of water.Although lateral roots were much thinner than nodal roots, the ratio of stele diameter to root diameter was almost the same in both (Figure 4(a)), suggesting that diameter of roots may determine their conductivity in both nodal and lateral roots.E. arundinaceus roots also have another morphological and anatomical traits adaptive for environmental stresses.The nodal roots, for example, have soil sheaths (Figure 3(a), Figure 3(b)) as in maize [39] [59].Dense root hairs of E. arundinaceus were observed in the soil sheath (Figure 3(a)), suggesting the involvement of root hairs with mucilage in the development of the soil sheath.Here we found that most parts along the root axis of E. arundinaceus nodal roots, including the mature parts, were tightly surrounded by the soil sheath, which may protect and help roots to absorb water and nutrients even in drought [28] [60].
The outer cortex of E. arundinaceus roots developed the hypodermis (Figure 3(c), Figure 3(d)).The hypodermis of grass roots can protect inner tissues including vascular systems from external biotic and abiotic stresses [26] [61].The hypodermis in the Erianthus root included partly multiseriate sclerenchyma with lignified cell walls as in rice roots [62] [63] and other cereals crops (e.g., pearl millet; [64]).The degree of sclerenchyma development can correspond to the drought tolerance ability of the plant.For example, several upland rice varieties with high drought tolerance form multiseriate sclerenchyma, whereas most lowland and many of drought-sensitive upland rice varieties possess single-layered sclerenchyma [65].Drought-tolerant sorghum cultivars develop thick sclerenchyma cell walls in contrast to drought-sensitive cultivars [29].Therefore, the formation of multiseriate and well-lignified sclerencyma in Erianthus roots may also contribute to high tolerance to environmental stress.Lateral roots of Erianthus, on the other hand, had no hypodermis (Figure 3(i)), which suggests their high permeability to water and nutrients in comparison with their parental nodal roots.
Matsuo et al. [16] already reported the formation of aerenchyma in the root cortex of submerged Erianthus in Thailand.Such aerenchyma was well developed in most nodal roots of E. arundinaceus thougheven in the not-submerged plants in this study (Figure 3(b), Figure 3(e)).This fact suggests that Erianthus may have adapted to tropical and subtropical monsoon climates where wet and dry conditions occur periodically.
Additionally, stele of Erianthus roots was characterized by multiseriate pericycle and accumulation of starch grains (Figure 3(f)-(g)).The lignified marginal cylinder of multiseriate pericycle with oval cells in stele is quite different from the single-layer pericycle of cereal crops (Figure 3(f)), but Raechal and Curtis [66] found sclerified multiseriate pericycles in some of Bambusoideae species.The starch grains accumulated could disappear in next summer.Yamashita and Okamoto [67] reported that numerous starch grains accumulated around secondary xylem of roots of mountain azalea (Rhododerndron Kurume group) in winter (December-February) and disappeared in summer (June-August).In Erianthus, adventitious lateral root are rarely formed, but the starch stock in roots may be advantageous for vigorous root activity under cold conditions in winter.Moreover, starch storage in winter may contribute to shoot regrowth in spring.

Concluding Remarks
Erianthus is one of the possible candidates for raw material crop for lignocellulosic bioethanol, because it has vigorous biomass production and high tolerance to environmental stresses.One of the reasons why Erianthus species have high performance is probably related to root system development.Large and deep root system has advantage for dry and poor soil conditions.Several anatomical traits including development of soil sheath including dense root hairs, hypodermis, aerenchyma, vascular system and starch grain accumulation characteristic in Erianthus could also be adaptive environmental stresses.

Figure 1 .
Figure 1.Erianthus root systems (a) and individual nodal roots with laterals (b) at 148 days after planting.Scale bars indicate 30 cm (a) and 10 cm (b).

Figure 2 .
Figure 2. Distribution of root length as a function of their diameters; the roots were collected by the monolith method at 80 (a) and 141 days (b) after planting.The numbers on the right of individual bars indicate the branching index.The error bar indicates standard error of total root length.

Figure 3 .
Figure 3. Anatomy of a Erianthus nodal (a)-(h) and lateral roots (i).Freehand cross-sections of nodal roots were taken at 5 cm from the base of the roots.The red color of the cell wall indicates lignifications detected by phloroglucinol staining (b), (c) (d) (f) and (i).Potassium iodide staining (g and h) detected starch in the stele of only Erianthus nodal roots in winter; the starch was stained black (g).Erianthus roots harvested in summer (h) had no starch in the stele.ae, aerenchyma in the mid-cortex; en, endodermis; ep, epidermis; hp, hypodermis; lx, large xylem vessels; lr, lateral root; ov, oval cell; ph, phloem; pt, parenchymatous cells in the stele; rh, root hair; sc, sclerenchyma; ss, soil sheath; and xy, small xylem vessel.The scale bars indicate 2 mm (a), 500 μm (b) and (h), 200 μm (c) and (e) and 100 μm (d) (f) and (g).

Figure 4 .
Figure 4. Relationship between root diameter and stele anatomical properties at 5 cm from the base of the nodal root.(a) Stele diameter; (b) Numbers of xylem poles (small xylem points) and large xylem vessels; (c) Mean diameter of large xylem vessels (LXV); and (d) Equivalent diameter of xylem vessels (XV).Linear regression line and correlation efficiency in (a) were calculated for nodal roots thinner than 3 mm.*** indicate significant correlation at p < 0.001.
lateral roots was similar to that in nodal roots (open squares in Figure4(a)).

Table 2 .
Characteristics of Erianthus roots collected by the monolith method at different growth stages (80 and 141 days after planting [DAP]).

Table 3 .
Correlation efficiencies of root diameter and stele properties at 5 cm from the base of nodal roots.Correlation efficiencies were calculated on the basis of data acquired from 21 nodal roots (d.f.= 19).*, **, and *** indicate significant correlation at p < 0.05, p < 0.01, and p < 0.001, respectively.PN, pole number; LXV, large xylem vessel; No., number; Σ(LXV area), total cross-sectional area of large xylem vessel elements; Eq XV diameter, equivalent xylem vessel diameter.