Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the
assimilation of ammonium by Triticum aestivum L.
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María
Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio
Irigoyen, Berta Lasa, Pedro María Aparicio-Tejo
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta
Lasa, Pedro María Aparicio-Tejo
Institute of Agri-Biotechnology, Public University of Navarre- CSIC-Government
of Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Spain.
Ignacio Irigoyen
Department of Agrarian Production. Public University of Navarre. Pamplona E31006, Navarre, Spain.
María Garnica, Fabrice Houdusse, José María Garcia Mina
Departament of R + D, TimacAgro-Grupo Roullier, Polígono ArazuriOrcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Spain.
Correspondence:
Pedro M. Aparicio Tejo
Instituto de Agrobiotecnología. Universidad Pública de Navarra-CSIC-Gobierno
de Navarra. Campus de Arrosadía E-31006 Pamplona, Navarra.Spain
Phone: +34 948169122
Fax: +34 948 232191
Email: pmapariciotejo@unavarra.es
http://www.agrobiotecnologia.es/en/grp-fisiologiaMolecular/index.htm
1
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the
assimilation of ammonium by Triticum aestivum L.
Abstract
The use of urea as an N fertilizer has increased to such an extent that it is now
the most widely used fertilizer in the world. However, N losses as a result of
ammonia volatilization lead to a decrease in its efficiency, therefore different
methods have been developed over the years to reduce these losses. One of
the most recent involves the use of urea combined with urease inhibitors, such
as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the
hydrolysis of urea in the soil. The aim of this study is to perform an in-depth
analysis of the effects that NBPT use has on plant growth and N metabolism.
Wheat plants were cultivated in a greenhouse experiment lasting four weeks
and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062,
0.125%). Each treatment was replicated six times. A non-fertilized control was
also cultivated. Several parameters related with N metabolism were analysed at
harvest.
NBPT use was found to have visible effects, such as a transitory yellowing of
the leaf tips, at the end of the first week of treatment. At a metabolic level,
plants treated with the inhibitor were found to have more urea in their tissues
and a lower amino acid content, lower glutamine synthetase activity, and lower
urease and glutamine synthetase content at the end of the study period,
whereas their urease activity seemed to have recovered by this stage.
2
Keywords:
ammonium
metabolism;
N-(n-butyl)
thiophosphoric
triamide
(NBPT); urea; urease; urease inhibitor; wheat.
Introduction
The widespread contamination arising from agricultural activities is currently
one of the most serious environmental problems in many European countries.
Indeed, the environmental problems associated with the use of N fertilizers
have led to a compromise between achieving increased productivity and a
quality harvest, and limiting their environmental impact. “Sustainable”
agricultural production requires an in-depth understanding of the processes
associated with the transformation of N in the soil and the different ways in
which it is absorbed by the plant, and how these factors affect the plant's growth
and development. The development of new fertilizers and new methods of
application, especially the use of products that can inhibit the activity of those
microorganisms associated with transformation of the N present in the soil, is
also important.
The use of urea as an N fertilizer has increased to such an extent in the last 2530 years that nowadays it is the most widely used form of N fertilizer in the
world (IFA 2009). The main advantages of this compound are its high N content
(46%), its relatively low cost, and its ease of use. However, the efficiency of
urea is markedly reduced due to N losses caused by, amongst other factors,
ammonia volatilization, which can result in the loss of more than 50% of the N
applied (Terman 1979; Bremner 1995). This occurs when urea is hydrolyzed by
the enzyme urease released by microorganisms in the soil to form ammonium,
3
which, depending on the pH of the soil, may subsequently be transformed into
highly volatile ammonia (Bremner and Krogmeier 1988).
Besides adapting normal agricultural practices for the incorporation of urea into
the soil, for example irrigation or by adjusting the dose and application time,
several strategies have been developed to reduce the losses resulting from
ammonia volatilization. Slow-release fertilizers, where the granules are coated
with relatively insoluble substances in order to delay the release of urea, and
the application of urease inhibitors, are the most widely used methods in this
respect (Trenkel 1997). Urease inhibitors, which have been the subject of
numerous studies in the last few years, interact with the active site of the
enzyme or a key functional group in the molecule to produce a change in the
structure of the active sites, thereby delaying urea hydrolysis and, in turn,
promoting the formation of ammonium (Watson 2000). However, a compound
has to meet certain requirements, such as lack of toxicity to the plant or the
environment, stability, efficiency at low concentrations, compatibility with urea
and a price that makes its use economically viable, to be considered a good
candidate as an agricultural urease inhibitor. Indeed, although numerous
substances have been evaluated, very few have been found to be really
effective.
The compounds tested include structural analogs of urea, phosphoramides and
thiophosphoramides, and hydroquinones and benzoquinones. N-(n-butyl)
thiophosphoric triamide (NBPT) is currently considered to be the most effective
means of controlling urea hydrolysis (Bremner and Chai 1986) as its oxidised
form, NBPTO, has a higher urease inhibition capacity (Creason et al. 1990).
Indeed, the majority of both laboratory and field studies have been carried out
4
with NBPT. This compound has been studied in different species, mainly
cereals (Bremner 1995; Malhi et al. 2001; O´Donovan et al. 2008), and has
proved to be effective in reducing ammonia volatilization and improving
agricultural yields (Bremner and Krogmeier 1988). In fact, it was the first urease
inhibitor to be marketed, under the commercial name Agrotain®.
Plants grown with urea and NBPT have been found to have a higher urea
content in their tissues (Watson and Miller 1996). This accumulation of urea
could be the cause of the phytotoxicity observed in plants grown with urea and
urease inhibitor (Krogmeier et al. 1989), and could therefore be a major
disadvantage of the use of this product. The majority of this urea is absorbed by
the roots, although some of it is endogenous, originating from the different N
metabolism processes in the plant. To the best of our knowledge, no studies
have yet focussed on the influence of NBPT absorption by plant roots and how
this absorption could inhibit plant urease and cause physiological changes in N
metabolism. The aim of the present study was therefore to evaluate the
effectiveness of NBPT at lower doses than those recommended commercially
and to study its effect on the plant's N metabolism.
Materials and methods
Growth conditions
Wheat seeds (Triticum aestivum L.) cv. “Fiel” were sown in 2 L pots containing
soil and perlite (1:1 v/v). The soil, with an alkaline pH (8.2), 1.5% organic matter
content and sandy loam texture, was obtained from Mendigorría (Navarre,
Spain) and was dried in air and sieved beforehand through a 2 mm mesh. A
urea solution (180 kg N ha-1) was applied to all pots along with different
5
concentrations of N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0.062% and
0.125% (w/w)). Additional phosphorous, potassium and magnesium were also
applied to the plants at doses of 100, 200 and 50 kg ha-1, respectively. Control
pots with no fertilizer applied were also prepared. Each treatment was
replicated six times. All 30 pots were placed in a greenhouse, where they
remained until the end of the experiment. The plants were irrigated with distilled
water up to field capacity during the four weeks that the study lasted whilst
ensuring that no drainage occurred.
Plant shoot growth and internal contents
The chlorophyll content (SPAD) and dry weight of plant shoots were measured
by placing the leaves in a hot air oven for 48 h at a temperature of 75 ºC. The
urea content was determined using a modification of the method described by
Witte et al. (2002), and using the colour reagent described by Kyllingsbaek
(1975). To avoid possible interference by other molecules in the urea
measurement, the samples were previously passed through anion-cation
exchange columns (Water Oasis MCX and MAX) to remove any ions that could
interfere with the analysis. Extraction of the soluble ions from the tissues was
performed in water at 80 ºC. The ammonium concentration was determined
using the supernatant obtained after centrifugation, using the phenolhypochlorite method (Solorzano 1969). Amino acids were extracted into 80%
ethanol at 80 ºC. After evaporation of the ethanol, they were re-suspended in
water and the amino acid content determined by the ninhydrin reaction using
glycine as a standard (Yemm and Cocking 1955). The soluble protein was
measured as described by Bradford (1976).
6
Enzyme activities
Urease activity in the plant tissues was determined by measuring the amount of
ammonium formed during the hydrolysis of urea. For the extraction, the sample
was ground in liquid N2 and homogenized using 50 mM phosphate buffer at pH
7.5 containing 50 mM NaCl, 1.5% PVPP (w/v), 1 mM EDTA, 2 mM DTT and 4
mM PMSF. After centrifuging the extracts (20 min, 13000g), the supernatants
were purified through columns (Sephadex G25-fine). The reaction was started
by the addition of 5 M urea, and was carried out at 50 ºC. Aliquots were taken at
0, 20, 40 and 60 minutes, and the ammonium generated was measured using
the phenol-hypochlorite method. The urease activity was calculated from the
standard curve obtained from the ammonium concentrations over time.
Glutamine synthetase (GS) was determined following the method of O’Neal and
Joy (1973).
Urease and glutamine synthetase (GS) contents
Urease and GS contents were assessed by Western blotting. The previously
described protein extracts used for the determination of the enzyme activities
were boiled for 2 minutes after adding the sample loading buffer described by
Laemmli (1970). SDS gel electrophoresis was then performed in discontinuous
gels, with 10% (w/v) polyacrylamide in the resolving gel and 4.6% in the
concentrator, at 100 mV. Molecular markers were also loaded (Biorad 1610305) in order to estimate the size of the polypeptides. After separation, the
proteins were transferred according to the method described by Towbin et al.
(1979), but using polyvinylidene difluoride (PVDF) membranes. The membranes
were then blocked by submerging them in a solution containing non-fat dry milk
7
(5%) after repeated washing with TTBS (45 minutes). Monospecific polyclonal
antibodies were used to estimate the urease and GS contents. For the GS
antibody, a synthetic peptide against a conserved zone of the GS gene was
used to immunize rabbits, as described by Cruz et al. (submitted for
publication). For the urease antibody, a purified urease from jack bean (SigmaAldrich) was used to immunize rabbits. The monospecific fraction (IgG) of the
serum was isolated in a column containing the antigen for both antibodies.
Incubation with the corresponding primary antibody at a 1:1000 dilution for the
rabbit anti-jack bean urease IgG, and 1:2000 for the rabbit anti-GS IgG, was
carried out overnight in TTBS at 4 ºC. Incubation with the secondary antibody
was performed for 1 h using a goat anti-rabbit antibody conjugated with
horseradish peroxidase.
Finally,
detection
was accomplished
using a
commercial chemoluminescence kit (Amersham ECL Plus, AmershamPharmacia).
Statistical analysis
The data obtained from the different analyses were subjected to variance
analysis (ANOVA), and the least significant difference (LSD) test was applied.
Different letters were assigned for significant inter-treatment differences
detected using the F-Fisher test. Regression analyses were performed between
NBPT (%) applied and different plant parameters. The control treatment was
excluded from these regressions. The SPSS statistics program was used to
perform all calculations, and a result was considered significant at a significance
level of 95%.
8
Results
Plant Growth
Dry weight showed a tendency to increase with NBPT inhibitor dose, with this
increase being significant at highest NBPT inhibitor dose (Fig. 1). The SPAD
values, which were used as indicators of the chlorophyll content, showed no
significant differences between plants grown with the different concentrations of
NBPT applied and those grown with urea but without NBPT. The non-fertilized
control plants showed visual symptoms of chlorosis, as confirmed by their low
SPAD values (Fig. 2). Seven days after application of the treatments, a slight
yellowing of the leaf tips, which increased in the following days, could be seen
in those plants treated with NBPT. The affected area increased with increasing
inhibitor dose (not shown), although this effect was only transitory since new
leaves showed no yellowing. Indeed, this phenomenon was hardly noticeable at
the end of the study. The yellow leaf tips were sampled separately in the
treatment with highest NBPT dose to determine their urea and ammonium
contents.
Internal contents of urea, ammonium, amino acids and soluble protein
The urea content in the leaves of the plants fertilized with urea increased with
amount of inhibitor applied in a dose-dependent manner (Fig. 3). No significant
differences were observed between the treatments with 0.012% and 0.062%
NBPT. The urea content of plants treated with urea but no inhibitor was similar
to that in the control plants. Those leaf tips that showed chlorosis had a urea
content of 1600 μmol g-1 DW, about 2000 times higher than the leaves of plants
fertilized with urea but no NBPT. In contrast, the plants fertilized with urea but
9
no NBPT had the highest ammonium content, whereas the control plants had
the lowest levels of NH4+ in the leaf (Fig. 4). The NH4+ content in those leaf tips
that showed chlorosis was slightly higher than in the plant leaves treated with
urea but no inhibitor (18.7 mol g-1 DW). The amino acid content of the nonfertilized control plants was lower than in the rest of the treatments. The plants
that received medium to high doses of inhibitor (0.062% and 0.125% NBPT)
had significantly lower leaf amino acid contents than those that received the
lowest dose or were fertilized with urea only (Fig. 5).
Differences in the leaf soluble protein content were also observed (Fig. 6).
Thus, the plants treated with an NBPT dose of 0.012% had the highest protein
values, with these values decreasing as the inhibitor dose increased. The
protein content of the plants fertilized with urea only was similar to that for the
plants that received the highest dose of inhibitor. A four- to fivefold decrease in
protein content was observed in the leaves of non-fertilized control plants.
Enzyme activities and content
The control plants had the highest urease activity values (Fig. 7). Urea appears
to inhibit urease activity in leaves, and the NBPT inhibitor induced no significant
changes in urease activity. Leaf glutamine synthetase (GS) activity was highest
in the control plants. For the plants fertilized with urea, only the highest doses of
NBPT (0.062 and 0.125 %) had an effect on the GS activity (Fig. 8).
The urease content, as estimated by Western blotting with a novel specific antijack bean urease antibody, was found to be higher in control plants and in those
treated with urea and low NBPT doses. Plants treated with the highest inhibitor
doses showed the lowest urease contents of all treatments (Fig. 9A). As for the
10
pattern of GS isoenzyme expression, two isoforms of leaf GS were detected: a
chloroplastic (GS2) isoform with a molecular weight of 45 kDa, and a cytosolic
(GS1) isoform with a slightly lower molecular weight of 40 kDa. The band
corresponding to the chloroplastic isoform was less intense in the leaves of the
control plants. Plants treated with urea and 0.125% NBPT showed a lower
intensity of the cytosolic isoform. The GS1 and GS2 levels were similar in the
other treatments.
Regression analysis
Regression analyses were performed using the data for all plants grown with
different NBPT doses to produce different curves. Both non-linear and linear
adjustments were found to give similar results, therefore all data were treated
with a linear regression analysis. The linear regression parameters can be
found in Table 1. The P-value is statistically significant for the parameters dry
weight, urea content, amino acid content and GS activity. These parameters
also presented the highest r2. The response to increases of NBPT dose was
positive for dry weight and urea content and negative for amino acid content
and GS activity.
Discussion
Effects on the biomass of wheat plants grown with urea in conjunction with the
urease inhibitor NBPT were only evident at the inhibitor dose recommended by
the manufacturer (0.125% w/w). Similar results were observed by Watson and
Miller (1996) in rye grass, although the growth period in this case was 10 days
and the doses used were 0.01%, 0.1% and 0.5% (w/w). The urea applied with
11
no inhibitor seems mainly to be hydrolyzed by urease in the soil in a process
that generates endogenous levels of urea in those plants fertilized with urea
similar to those in the control plants (non-fertilized plants). The N deficiency is
demonstrated by leaf chlorosis and the slower plant growth, as well as by other
effects typically observed in the control plants. The soil employed in the
experiment exhibited a low N content that was insufficient to enable plants to
develop normally. This N deficiency meant that the plant metabolism was
affected, as observed by the lower amino acid and protein contents in the nonfertilized plants.
It should be pointed out that the activity and content of GS (particularly cytosolic
GS1), a key enzyme in the assimilation of N, were highly induced in nonfertilized control plants, possibly with the aim of assimilating this element with
the highest possible efficiency if its availability in soil increased. The urease
activity in the control plants was also significantly higher than in the rest of the
treatments. The urease protein content was similar to that in other treatments,
except at the highest inhibitor dose. Furthermore, no contribution from urea was
observed for the control plants, hence the urease activity in this case could
arise from the remobilization of N-compounds, as occurs during the seed
germination process (Zonia et al. 1995). At the end of the study (4 weeks),
NBPT showed no obvious effects on urease activity. Several authors have
described a temporary urease inhibition lasting for approximately 10 days after
the application of NBPT (Watson and Miller, 1996), although this period varied
according to the concentrations of NBPT used. However, Krogmeier et al.
(1989) reported no urease inhibition in wheat and sorghum 21 days after
applying the inhibitor. These authors attributed this effect to recovery of the
12
urease or the degradation of NBPT during those three weeks. In our study,
temporary urease inhibition may well explain the transient yellowing of the leaf
tips at day 7 after treatment, with the plants again being capable of forming
healthy leaves soon after. In previous studies performed by our group
(Cruchaga et al. 2006), yellowing of the edges of the leaves was observed in
peas treated with NBPT although, as in the present study, the new leaves
developed normally. Watson and Miller (1996) also noted a temporary yellowing
in their study with rye grass. Although urease activity inhibition was not
observed at the end of the study, urease content seemed to be affected only by
the highest concentration of NBPT. The higher urea concentration in plants
treated with NBPT led us to consider that both leaf and soil urease were
inhibited during the initial days of the study, thereby allowing more urea to
accumulate in the plant. This may explain the dose-dependent effect of NBPT
on urea content observed by Watson and Miller (1996) and Krogmeier et al.
(1989). Our results seem to indicate that this yellowing is not due to ammonium,
as suggested by other authors (Bremner et al. 1989), but to the accumulation of
urea, as described by Krogmeier et al. (1989), since the NH4+ concentration in
the leaf tips during the yellowing was similar to that in the non-yellow leaves of
plants treated with urea only. A possible cause of this symptom could be the
accumulation of toxic levels of urea in the leaf tips. All plants were, however,
able to recover and form new healthy leaves. Lower ammonium contents were
expected in the plants treated with NBPT since urea degradation either did not
take place or occurred to a lesser extent. After one month of growth, leaf urease
was no longer inhibited and the ammonium concentration was similar in all
NBPT treatments, although lower than in plants treated with urea only. In
13
contrast, GS activity seems to be affected negatively by NBPT, which could
reduce the amino acid content by increasing the inhibitor dose. Taken together,
these results seem to indicate that one month after applying NBPT, the effects
of this compound on the activity of urease have either been reversed or
compensated, leaving the accumulation of urea and the decrease of GS activity,
amino acid content and urease and GS2 content in leaves at the highest NBPT
concentration as the only signs of the initial urease inhibition. On the other
hand, the soluble protein content is a parameter that initially responds to N
availability, which shows that treatment with 0.012% and 0.062% NBPT
improves the N availability. In these cases, the endogenous levels of urea are
higher than for the control treatment but lower than for the 0.125% NBPT
treatment, whereas the urease content for the 0.125% NBPT treatment is lower
than for the other treatments, thus suggesting that it could temporarily affect N
availability and hence protein content.
Thus, when NBPT is applied to the soil, particularly at high doses, it is absorbed
by the plant and modifies its metabolism, although the effects are only
transitory. Future experiments will therefore be required to determine the
agricultural advantages of fractionated application of both fertilizer and inhibitor
at doses lower than those recommended by the manufacturer.
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17
0,4
a
ab
0.012%
0.062%
-1
(g plant )
0,3
Dry weight
ab
b
0,2
c
0,1
0,0
control
0%
0.125%
UREA + NBPT
Fig. 1 Dry weight of wheat plants grown for four weeks without (control
treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
Values are means (n=6) ± SE.
50
a
a
a
a
0%
0.012%
0.062%
0.125%
SPAD
40
30
b
20
10
0
control
UREA + NBPT
Fig. 2 The SPAD values of wheat plants grown for four weeks without (control
treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
Values are means (n=6) ± SE.
18
a
b
2
b
-1
(µmol g DW)
Urea content
3
1
c
c
control
0%
0
0.012%
0.062%
0.125%
UREA + NBPT
Fig. 3 Leaf urea content of wheat plants grown for four weeks without (control
treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
Values are means (n=6) ± SE.
(µmol g DW)
a
-1
Ammonium content
20
15
b
10
b
b
c
5
0
control
0%
0.012%
0.062%
0.125%
UREA + NBPT
Fig. 4 Leaves ammonium content of wheat plants grown for four weeks without
(control treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
Values are means (n=6) ± SE.
19
a
a
100
b
80
b
-1
(µmol gly g DW)
Free amino acid content
120
60
c
40
20
0
control
0%
0.012%
0.062%
0.125%
UREA + NBPT
Fig. 5 Total amino acid content in leaves of wheat plants grown for four weeks
without (control treatment) and with N-fertilization of 180 kg urea-N ha-1 at four
NBPT concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to
urea). Values are means (n=6) ± SE.
200
a
b
c
c
120
-1
(mg g DW)
Protein content
160
80
40
d
0
control
0%
0.012%
0.062%
0.125%
UREA + NBPT
Fig. 6: Leaf protein content of wheat plants grown for four weeks without
(control treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
Values are means (n=6) ± SE.
20
a
3
-1
(mU min mg protein)
2
-1
Urease activity
4
1
b
b
0%
0.012%
b
b
0
control
0.062%
0.125%
UREA + NBPT
Fig. 7 Urease activity of wheat plants grown for four weeks without (control
treatment) and with N-fertilization of 180 kg urea-N ha-1 at four NBPT
concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to urea).
-1
-1
( mol GHM min mg protein)
GS activity
Values are means (n=6) ± SE.
0,30
0,25
a
0,20
0,15
b
0,10
bc
c
d
0,05
0,00
control
0%
0.012%
0.062%
0.125%
UREA + NBPT
Fig. 8 Glutamine synthetase (GS) activity of wheat plants grown for four weeks
without (control treatment) and with N-fertilization of 180 kg urea-N ha-1 at four
NBPT concentrations (0%, 0.012%, 0.062% and 0.125% w/w with respect to
urea). Values are means (n=6) ± SE.
21
Urea + NBPT
Control
0%
0.012% 0.062% 0.125%
A
90 kDa (Urease)
B
45 kDa (GS2)
40 kDa (GS1)
Fig. 9 Urease (A) and glutamine synthetase (B) content of wheat plants grown
for four weeks without (control treatment) and with N-fertilization of 180 kg ureaN ha-1 at four NBPT concentrations (0%, 0.012%, 0.062% and 0.125% w/w with
respect to urea). This Western blot was repeated 6 times, with similar values
being obtained on each occasion.
Table 1 Values of the coefficients a and b in the linear regression equation y =
a x + b (y = plant parameter; x= percentage of NBPT). Standard Errors of the
coefficients, regression coefficient (r2) and P-value of the adjustment are
presented (n = 24).
Plant parameters
Dry weight
SPAD
Urea content
Ammonium content
Aa content
Soluble protein
Urease activity
GS activity
a
0.3
4.8
11.1
-10.6
-278.1
-84.3
-1.7
-0.4
Standard
error of a
0.14
6.66
1.87
8.59
63.30
63.67
1.11
0.04
b
0.3
40.7
1.1
13.5
98.6
131.5
0.7
0.1
Standard
error of b
0.01
0.47
0.13
0.58
4.44
4.47
0.08
0.00
r2
0.22
0.02
0.61
0.07
0.47
0.07
0.12
0.81
P-value
0.02
0.48
0.00
0.23
0.00
0.20
0.15
0.00
22
23