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Dep. of Plant Sciences, Univ. of Rhode Island, Kingston, RI 02881 USA
senmike{at}uri.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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0.05) and positively correlated with the total biomass, length and area of the belowground organs. Fibrous roots contributed to > 80% of the total belowground length. Approximately 80% of the total fibrous root length had diameters < 0.2 mm. The fibrous root length, surface, and volume of every diameter class were significantly and positively correlated with NUR. Larger numbers of thick roots (diameter 
0.5 mm) appeared to have no effects on NUR, while increased rhizome number appeared to have a negative effect on NUR.
Abbreviations: NUR, nitrate uptake rate
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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While nitrate uptake by turfgrasses has been studied extensively in the past decade (Bowman et al., 1989; Cisar et al., 1989; Liu et al., 1993; Jiang and Hull, 1998a, b), few studies have examined the relationship between nitrate uptake and root morphological characteristics in turfgrasses. In a study comparing nitrate leaching losses from seeded vs. sodded Kentucky bluegrass turfs, Geron et al. (1993) suggested that greater root mass contributed to less nitrate leaching under the seeded turf due to increased nitrate uptake. Bowman et al. (1998) examined the effect of root architecture on nitrate leaching and found that nitrate leaching from a shallow-rooted genotype of creeping bentgrass (Agrostis palustris Huds.) was greater than from a deep-rooted genotype. However, the shallow-rooted genotype had a higher NUR than the deep-rooted genotype, expressed on a root weight basis. They suggested that the shallow-rooted genotype may have finer roots, which would produce a larger absorption area per unit weight.
Root development appears to be genetically determined and mutations affecting root initiation, meristematic activity, and radial structure have been described (Rost and Bryant, 1996). Large genotypic differences in root mass distribution have been demonstrated in bermudagrass [Cynodon dactylon (L.) Pers.], buffalograss [Buchloë dactyloides (Nutt.) Engelm.], creeping bentgrass, Kentucky bluegrass, perennial ryegrass (Lolium perenne L.), red fescue (Festuca rubra L.), and zoysiagrass (Zoysia spp.) (Boeker, 1974; Ensign and Weiser, 1975; Hays et al., 1991; Marcum et al., 1995a, b; Salaiz et al., 1991). Further, parent-progeny regression of 10 creeping bentgrass clones has indicated high narrow-sense heritability values for root number and area at deeper soil profiles (Lehman and Engelke, 1991). If the relationship between root morphological traits and nitrate uptake can be established for these species, progress could be made toward genetic improvement in nitrate uptake capacity in turfgrasses.
This study was conducted to examine the relationship between belowground morphological characteristics and nitrate uptake capacity in Kentucky bluegrass cultivars. Our objectives were to determine (i) NUR by clonal plants of selected cultivars, as affected by nitrate deprivation, and N use efficiency in various parts of the plant; (ii) whether these cultivars could be differentiated on the basis of various root morphological traits; and (iii) the relationships between these morphological traits and NUR. Results concerning the second and the third objective are reported in the present paper and results concerning the first objective are reported separately.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Thirty days after seeding, seedlings were transferred to growth flats containing a soil-perlite mixture (1:1 by volume) with each seedling planted in an individual cell in March of 1998. Three and one-half months later, six tillers were separated from one seed-generated plant with the largest number of tillers. These tillers were planted in individual polystyrene containers (3.8 cm in diameter, 21 cm in depth, one bottom and four side drainage holes, Stuewe & Sons, Inc., Corvallis, OR) filled with silica sand. They were placed in a greenhouse and irrigated daily except for weekends with a one-half strength N-free nutrient solution (Hoagland and Arnon, 1950), supplemented with 1 m M NaNO3. During weekends, tap water was used. Grasses were mowed once every week at a 5-cm height. After 5 mo of growth and maintenance, tiller-generated plants completely filled their containers with five to 10 new tillers. Four of the six tillered plants of each cultivar were used for nitrate uptake determination and root morphological measurements.
Nitrate Uptake Measurement
Three days before nitrate uptake measurements, grasses were mowed at a height of 5 cm and irrigated with tap water the following day. One day before uptake measurements, plants were excavated from polystyrene containers and roots of each plant were gently washed under running tap water for 5 min to remove sand completely. The plants were then supported by cotton plugs, in individual 130-mL growth vessels with roots submerged in 120 mL of tap water, and transferred to a walk-in growth chamber. After 24 h in tap water (Day 1), each plant was supplied with 120 mL of a one-half strength Hoagland's nutrient solution containing 0.5 mM NaNO3. During solution replacement, roots were gently rinsed twice in 8 L of the same solution. The solution in each growth vessel was continuously aerated through an aeration line connected to an air pump via a manifold. The atmosphere of the growth chamber was circulated by a built-in electric fan. The day–night temperatures in the chamber were 24 and 15°C and the photosynthetic photon flux density at plant height was 800 µmol m-2 s-1 with a photoperiod of 14 h. After the plants had been in the nutrient solution for 24 h, a 2-mL sample of the nutrient solution was taken from each growth vessel and the volume of the remaining solution was recorded to determine nitrate depletion. The nutrient solution was replaced and the procedure was repeated each day over an 8-d period, except on Day 6, when tap water instead of nutrient solution was supplied to the plants.
Nitrate concentration of nutrient solutions was determined by a Rapid Flow Analyzer (RFA 300, ALPKEM Corp., Clackamas, OR) fitted with a rotary tray, which can hold 90 2-mL sample cups. Net nitrate uptake was calculated from nitrate depletion data and the rate was expressed as micromoles of nitrate absorbed per plant per day.
Root Quantification
After nitrate uptake measurements, the plants were separated into six parts: leaf blades, sheaths, dead but attached leaves (thatch), rhizomes, fibrous roots, and adventitious roots, which were newly generated during the 8-d uptake period. All plant parts except thatch were scanned by a scanner (ScanJet 4c, Hewlett-Packard Co.) contained in a Delta-T SCAN Splash Protection System (Delta-T Devices Ltd., Cambridge, England). Fibrous root systems were cut into five portions along the axis and root pieces were completely spread in Delta-T SCAN root handling dishes containing a 3-mm layer of deionized water. Water facilitated the dispersal and separation of fibrous roots. Other parts were spread in a dry dish. The dish, which has a transparent glass bottom, was placed on the scanner and covered with a light-proof, white cover. Because fibrous roots were brown, they were also scanned with a white background and without staining. Adventitious roots were scanned with a black background because of their white color. The fibrous root samples were spread and scanned three times and other parts were scanned once.
The scanning brightness was tested with a small sample of each plant part and selected for each so that the estimated width and length were closest to the real width and length of each sample. The scanner resolution was set at 400 dpi (dot per inch) so that fibrous roots with small diameters (0.0635 mm) could be detected without creating excessively large files. The scale was set at 100%, and images were recorded as black-and-white line drawings and saved in an uncompressed, tagged image file format to make the images compatible with the image analysis software.
A Delta-T SCAN image analysis software (Delta-T Devices Ltd.) was used to analyze the image files acquired above. The total length, total area (image area), and average thickness of a sample were calculated simultaneously by the Length analysis function based on a procedure described by Harris and Campbell (1989). In addition, image files of fibrous roots were analyzed by the Length Sin 
function, which estimates length distribution among specified thickness ranges by using trigonometric functions (Delta-T SCAN User Manual, Delta-T Devices Ltd., 1993). The total surface area and total volume in each thickness group were calculated by the Length Sin function, assuming that roots are stick-type objects.
After scanning, the fresh weights of all plant parts were recorded and after oven-drying at 70°C for 3 d, the dry weights were recorded. The specific length, surface area, and volume of the fibrous roots were calculated from the dry weight and total length, total surface, and total volume of each sample.
Data Analysis
The experiment was conducted in a randomized complete block design with four replicates. Cultivar means of NUR were grand means of all dates and replicates for each cultivar (7 x 4 or 28 observations). Cultivar means of fibrous root parameters were means of all three images and four replicates (12 observations). Data were analyzed by an ANOVA (analysis of variance) procedure and significant means were separated by the DUNCAN (Duncan's Multiple Range Test) procedure within the Statistical Analysis System (SAS for Windows v. 6.12, SAS Institute Inc., Cary, NC). Regression analysis was performed with NUR as the dependent variable and root parameters as independent variables by the Minitab statistical analysis software (Minitab for Windows rel. 12, Minitab Inc. State College, PA).
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Results and discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Among the six cultivars selected, Barzan, Eclipse, and Gnome showed the lowest NURs on a per-plant basis (Table 1) . Barzan and Gnome were also the poorest performers among the six cultivars in terms of turfgrass quality ratings in the NTEP trials (NTEP Final Report 1991–1995). One of the two best NTEP trial performers, Blacksburg, showed the highest NUR whereas the other best performer, Eclipse, exhibited a much lower uptake rate. Conni and Dawn were intermediate performers, as were their NUR rates (Table 1). These results appeared to suggest that NURs on a per-plant basis are closely related to field performance of turfgrass cultivars.
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0.05 (Table 1). Total length, total area, and average diameter of fibrous roots were significantly (P 0.05) greater in Blacksburg than in the other five cultivars. Although significant differences (P 0.05) in these fibrous root traits were not found among the other five cultivars, their rankings in relation to these parameters closely followed those for field turfgrass quality, with the exception of Eclipse (Table 1; NTEP Final Report 91–95). Adventitious roots in this study represented newly formed roots emerging during the uptake measurement period, and the traits of these roots should indicate the vigor of new root formation. Total length and area of adventitious roots were greater in Conni than in Barzan and Eclipse (Table 1). Significant (P 0.05) variation in rhizome formation was observed among the six cultivars, with Gnome producing few recognizable rhizomes. Among cultivars that produced substantial rhizomes, Barzan had greater rhizome length and area than Blacksburg and Conni while Dawn and Eclipse were intermediate. Simple correlation analyses documented an r of -0.69 for fibrous root length vs. rhizome length and an r of -0.67 for fibrous root area vs. rhizome area when excluding Gnome data. These results indicated a negative relationship between fibrous root production and rhizome production.
Fibrous roots constituted more than 80% of the total length of the belowground plant parts (Table 1). Because these roots are the organs that actually absorb the most nitrate from soil, distributions of their length, surface area, and volume among various diameter ranges were analyzed (Table 2)
. Averaged over all cultivars, fibrous roots with diameters <0.2 mm contributed 79, 53, and 24% to the total fibrous root length, surface, and volume, respectively, while fibrous roots with diameters 0.5 mm contributed 4, 15, and 40% to the total length, surface, and volume, respectively. Cultivar differences were found in the distributions of all three traits in every diameter range except that between 0.2 and 0.5 mm (Table 2). These medium roots showed no intraspecific differences in their contribution to the total length and surface, but in Blacksburg, medium roots contributed a lesser volume of its total fibrous root volume than they did in other cultivars (Table 2).
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0.05 (Table 3).
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The specific length of fibrous roots showed wide variation among cultivars, with Conni having the longest total root length per unit dry weight (Table 4) . Blacksburg and Eclipse had shorter specific root length than all other cultivars except Dawn. Specific surface and volume showed similar patterns of variation. These parameters could indicate the fineness, and biomass or carbon status of the root system, a higher value suggesting a finer root system but a lower biomass (Boot, 1989). However, in the present study, there was no relationship between root mean diameter and specific length, which may be due to the fact that total length is mainly determined by the number of fine roots, whereas mean diameter is affected more strongly by the thickest roots. Root carbon status has been shown to affect nitrate assimilation in the roots and N use efficiency of Kentucky bluegrass (Jiang and Hull, 1999) and should have an effect on turfgrass field performance. Blacksburg and Eclipse had shorter total length per unit dry weight (Table 4), indicating higher carbon levels in their roots. These two cultivars were also the best performers among the six cultivars in the 1991 to 95 NTEP trials (NTEP Final Report 91–95, 1996).
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0.01) and positive linear relationships with NUR by the whole plant (Table 5)
. Average diameter of the three belowground parts was not significantly correlated at P 0.05. When regression analysis was performed by individual belowground parts, all parameters of fibrous roots were significantly (P 0.001) and positively correlated with NUR, with length and area having the greatest R2 and F values (Table 5). Although adventitious roots eventually mature into fibrous roots, contrary to the observations in fibrous roots, fresh and dry weights of adventitious roots had stronger relationships with NUR (greater R2 and F values) than did their length and area (Table 5). These results should indicate the importance of biomass accumulation in an early stage and root extension in a later stage to the uptake of nutrients. Average diameter of adventitious roots did not show a significant relationship at P 0.05 level.
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0.1) but negative linear relationship was apparent between NUR and all individual traits except rhizome length (Table 5). When Gnome was included, no significant linear relationships were found between rhizome parameters and NUR but significant (P 0.05) quadratic relationships were observed between NUR and the fresh weight, dry weight, and total length of rhizomes (Table 5). An example of this relationship is shown in Fig. 1
. Gnome produced few recognizable rhizomes compared to the other five cultivars as indicated in Table 1. We observed that rhizomatous nodes produced some fibrous roots, which increased the total fibrous roots beyond those formed from crown tissues. We have also shown a negative relationship between fibrous root length and rhizome length with Gnome excluded (Table 1). Taken together, these results suggest that an optimum level of rhizome production is necessary for the maximum uptake of nitrate.
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0.001) and linearly correlated with nitrate uptake (Table 6)
. Length, surface, and volume of fibrous roots with diameters between 0.2 and 0.5 mm had highest R2 and F values, while those with diameter 1.0 mm had lowest R2 and F values (Table 6). Further regression analyses revealed that when diameter range was 1.0 mm, the relationship between fibrous root length and nitrate uptake took the form of a hyperbola rather than that of a straight line (Fig. 2)
. The results appeared to indicate that the formation of very large fibrous roots could increase nitrate uptake by the plant only to a certain point, after which no further increase in nitrate uptake could be achieved, whereas the production of medium and fine roots could increase nitrate uptake without apparent limits.
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We have shown that approximately 80% of total root length was contributed by roots with diameter <0.2 mm. Compared with field-grown corn, which had 7% of total length contributed by roots with diameter <0.2 mm (Dowdy et al., 1995), Kentucky bluegrass has a substantially finer root system (Table 2). If nitrate uptake occurs predominantly in thicker roots as indicated by Henriksen et al. (1992) and Siebrecht et al. (1995) in barley, the characteristic fine root system of Kentucky bluegrass may be one of the reasons that it is generally an inefficient N user in the field. However, finer roots could produce a larger surface area per unit of root mass than larger roots. This may be why Kentucky bluegrass showed a high uptake rate per unit fresh weight in a previous study (Jiang and Hull, 1998a).
We have found significant (P 0.05) intraspecific variation in rhizome formation (Table 1), which confirms an early observation by Ensign and Weiser (1975). We also indicated that the two belowground sink organs, fibrous roots and rhizomes, could compete for carbohydrate supply from the shoots, and produce different effects on plant nitrate uptake (Tables 1 and 5). This is possible because photosynthate translocation to belowground sink organs is very limited, less than 2% of fixed carbon being translocated to roots within 24 h (Hull, 1981; Jiang and Hull, 1999). However, a certain amount of rhizome growth may increase the potential of fibrous root formation at rhizomatous nodes and help the plant explore a large volume of soil for nitrate and other nutrients. As indicated in Fig. 1, only an optimum level of rhizome production may be necessary for maximum nitrate uptake. Selecting Kentucky bluegrass varieties that produce an optimum length of rhizome with respect to nitrate uptake may be a strategy for increasing its nitrate uptake.
Genes involved in root development have been studied in many plants, but no such studies in turfgrasses have come to our attention. Research in other plants has led to the identification of genes involved in the development of root cap, pericycle, lateral roots, epidermis, cortex, elongation zone, and root apical meristem (Rost and Bryant, 1996). In rice (Oryza sativa L.), quantitative trait locus analysis and single-marker analysis have demonstrated close associations of some quantitative trait loci with root thickness, root/shoot ratio, and root dry weight per tiller, and a lack of association with maximum root depth (Champoux et al., 1995). Since the importance of root size and morphology for the efficient uptake of nutrients has been demonstrated in many crops (Boot, 1989) and in Kentucky bluegrass by the present study (Tables 5 and 6), a second component of a genetic program aimed at improving nitrate uptake might logically be the study of genes involved in root morphological development.
A third component of such a genetic improvement program would be the study of genes encoding nitrate transporters. Genetic and molecular studies on nitrate uptake in Arabidopsis and other plants have led to the characterization of genes that encode a high-affinity and a low-affinity nitrate transporter (Wirén et al., 1997). In previous studies (Liu et al., 1993; Jiang and Hull, 1998b) and in the present study (data not shown), intraspecific differences in NUR expressed on a fresh weight or root length or surface area basis have been demonstrated. These differences could be attributable to differences in the expression of the genes encoding the high- and/or low-affinity nitrate transporters. From the above discussion, it can be concluded that the ability of a Kentucky bluegrass to absorb nitrate from the soil is probably controlled at several levels: the partitioning of energy between roots and rhizomes, the root morphology, and the nitrate transporters. Genetic improvement at these three levels could produce turfgrasses that absorb nitrate more efficiently.
In summary, we have demonstrated significant (P 0.05) differences among clonal plants of Kentucky bluegrass cultivars in a number of belowground morphological traits including root and rhizome systems, and root diameter distributions. We have also demonstrated significant (P 0.01) and positive relationships between root morphological traits and NUR by the plant and the impact of rhizome production on root formation. It has also been found that developmental stage, nutrient level, and environmental conditions all influence phenotypic variation in root morphology (Boot, 1989). These factors can be altered or taken into consideration when formulating turfgrass management strategies. A better understanding of how turfgrass management practices can affect root morphology and rhizome production could also lead to enhanced turfgrass nitrate uptake.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Received for publication May 15, 1999.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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