Effect of NH4, NO3, and Glycine on Biomass and Flowering of Two Types of Brassica rapa in
Monoculture and Mixed Pots
Introduction:
Most ecologists agree that nitrogen is one of the most important nutrients for growth and
flowering of plants, among other physiological processes. For many years it was thought that
plants could only take up inorganic forms of nitrogen efficiently from the soil. Classic
ecological thought on nitrogen cycling states that plants depend on microbes in the soil that fix
organic nitrogen into inorganic forms: NO3 and NH4. This classic paradigm, however, has been
called into question, and a new paradigm has been proposed whereby plants can also take up
organic monomers from the soil, rather than use solely NH4 and NO3 (Schimel and Bennett
2004, Appendix: Figure 5). Recent studies have shown that it is possible for plants to take up
amino acids such as glycine, alanine, glutamic acid, and aspartic acid as well as more complex
organic N sources (Nasholm et al. 2000). The nitrogen source, organic, NH4, or NO3, that will
be taken up by plants would depend on the environment that they are in, being dependent on
such factors as productivity and competition (Appendix: Figure 6). What forms of nitrogen that
a plant can take up from the soil could have important implications for its growth, including
biomass and reproduction; and also could make the plant a better competitor, both against other
species and within its own species.
Brassica rapa (rapeseed) is a fast-growing herb that has two varieties: tall and dwarf, and
it is generally found in low productivity systems. Although these two forms of rapeseed are not
distinct species, they may be sufficiently diverse to favor the use of different nitrogen sources
and allocate their resources differently to growth and reproduction. Plants that are grown in
close proximity would be forced to compete for limited nitrogen in the soil. The nitrogen source
available may impact the interspecific and intraspecific competition of tall and dwarf rapeseed,
resulting in different biomass and flowering responses. In order to test the hypothesis that N
source will affect the intra or interspecific competition of tall and dwarf rapeseed, each variety
was grown in monoculture and mixed pots with three varying nitrogen treatments applied:
glycine, NH4, and NO3.
Materials and methods:
Sand was obtained from a home supply store, and then autoclaved for 30 minutes at
200F on the dry cycle. The pots, which were 0.74L, were sterilized using a 5% bleach solution.
The sterilized sand was added to the pots. We obtained tall and dwarf Brassica rapa seeds from
Carolina Biological Supply. There were two mixture types: monoculture and mixed. In
monocultures, we planted 30 seeds of either tall or dwarf seeds; in the mix cultures, 15 seeds of
each type were planted. There were three treatments with varying nitrogen sources: glycine,
NO3, and NH4. Each form of nitrogen had an equal number of moles of nitrogen. A control
group was also grown without an added nitrogen source. There were 6 repetitions. We excluded
any individuals that did not survive from the analysis. We grew the plants in the Kalamazoo
College greenhouse for 22 days and watered them as needed. We added three doses of nitrogen
to appropriate pots during the experiment.
After 22 days, we harvested the plants. The number of stems of dwarf and tall rapeseed
was recorded, and the number of flowers of each type was also counted. The stems were clipped
at the soil level, and dried for 48 hours at over 80C. The plants, which had been separated into
dwarf and tall, were massed to determine the dry weight biomass. Biomass and flowering per
stem was calculated, and were used in statistical analysis. We used a two-way ANOVA to
analyze the data for biomass and flower number. To test the differences among the groups, we
used Fisher’s LSD.
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Results:
We observed that both tall rapeseed and dwarf rapeseed were able to take up all three
forms of nitrogen, glycine, ammonium, and nitrate, from the soil in order to grow. Although no
statistical analysis was done comparing the no-fertilizer pots with the fertilized pots, in all cases,
plants that had been fertilized grew more than those that had not been fertilized.
Tall rapeseed analysis
The biomass of tall rapeseed was significantly affected by the nitrogen source (p-value
0.004396, Table I). The biomass/stem was greater for plants grown with NO3 than either glycine
or NH4 (Figure 1). There was no difference between the biomass of tall rapeseed grown in NH4
or glycine. Inter/intraspecific competition (mix) did not have an effect on the biomass of tall
rapeseed; biomass was the same whether grown in monoculture or in mix. Also, the interaction
between inter/intraspecific competition and N source did not significantly affect the biomass of
tall rapeseed. Inter/intraspecific competition had a significant affect on flowering (p = 0.02644).
When tall rapeseed was grown in monoculture, there were more flowers/stem than when tall
rapeseed was grown with dwarf rapeseed (Table II, Figure 2). Flowering of tall rapeseed was not
affected by N source or the interaction between N source and mix.
Table I. Analysis of variance of the biomass of the tall rapeseed
N source has a significant effect on biomass of tall rapeseed. There are three N sources: glycine, NH4,
and NO3. The mixes are monoculture and mix of tall rapeseed and dwarf rapeseed. R2 equals 0.582635.
Mix effect was not significant, nor was the interaction between mix and N source.
Source
N source
Mix
N sourceMix
Error
SS
df
MS
F-ratio
P-value
0.000018 2 0.000009 6.538848 0.004396
0.000002 1 0.000002 1.724613 0.199057
0.000001 2
0
0.307641 0.737471
0.000041 30 0.000001
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Figure 1. Effect of nitrogen source on the biomass of tall rapeseed
The biomass per stem of the NO3 treatment had a significantly higher biomass than glycine and NH4
treated plants, represented by A and B, with a p-value of 0.004396. Biomass was measured in grams.
The error bars represent ±1 standard error of the mean.
Table II. Analysis of variance of flowering of the tall rapeseed
Mix, monoculture or mixed pot, has a significant effect on the flowers/stem of tall rapeseed. The mix
represents the interspecific or intraspecific competition. There are three N sources: glycine, NH4, and
NO3. The R2 is 0.58141. N source effect was not significant, nor was the interaction between N source
and mix.
Source
N source
Mix
N source*Mix
Error
SS
df
MS
F-ratio P-value
0.77496 2 0.38744 2.33206 0.11445
0.90567 1 0.90568 5.45082 0.02644
0.86482 2 0.43247 2.60249 0.09073
4.98458 30 0.16615
Figure 2. Effect of inter/intraspecific competition on flowering of tall rapeseed
The mix and monoculture treatments are statistically different, represented in the figure as A
(Monoculture), B (Mix). There is greater flowering in monoculture than in mix. Flowering was
quantified as the average number of flowers per stem. The error bars represent ±1 standard error of the
mean.
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Dwarf rapeseed analysis
The biomass of dwarf rapeseed was significantly affected by inter/intraspecific
competition (p –value = 0.0366, Table III), but was not significantly affected by the N source or
the interaction between inter/intraspecific competition and N source. Dwarf rapeseed grown in
monoculture had a greater biomass/stem than those in a mix (Figure 3). Flowering of dwarf
rapeseed was significantly affected by the interaction between inter/intraspecific competition and
N source; dwarf rapeseed grown in a mix had higher flowering when grown in NO3 (p-value =
0.0301, Table IV). There was no difference in flowering among dwarf rapeseed in monoculture;
additionally, mixed dwarf rapeseed had equal flowers/stem in glycine and NH4.
Table III. Analysis of variance of biomass of dwarf rapeseed
The mix had a significant effect on the biomass of dwarf rapeseed. The mixes are monoculture and mix
of tall rapeseed and dwarf rapeseed. There are three N sources: glycine, NH4, and NO3. R2 is 0.48287. N
source and the interaction between N source and mix did not have a significant effect on biomass of dwarf
rapeseed.
Source
N source
Mix
N source*Mix
Error
SS
df
MS
F-ratio P-value
0.00002 2 0.00001 1.12714 0.33729
0.00004 1 0.00004 5.01732 0.03266
0.00002 2 0.00001 0.92506 0.40752
0.00026 30 0.00001
Figure 3. Effect of inter/intraspecific competition on biomass of dwarf rapeseed
Biomass of dwarf rapeseed was greater in monoculture than in mix. Biomass was quantified as the
average of the biomass per stem. There is a significant difference between mix and monoculture, as
shown by A and B in the figure. The error bars represent ±1 standard error of the mean.
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Table IV. Analysis of variance of flowering of dwarf rapeseed
The interaction between N source and mix had a significant effect on flowering of dwarf rapeseed. There
are three N sources: glycine, NH4, and NO3. The mixes are monoculture and mix of tall rapeseed and
dwarf rapeseed. R2 is 0.58454. The N source did not have a significant effect on the flowering.
Source
N source
Mix
N source*Mix
Error
SS
df
MS
F-ratio P-value
0.0240 2 0.0240 0.2000 0.6578
0.8988 1 0.4494 3.7420 0.0354
0.9471 2 0.4735 3.943 0.0301
3.6028 30 0.120
Figure 4. The effect of nitrogen source and inter/intraspecific competition on flowering of
dwarf rapeseed
There was a significant increase in flowering of the mixed dwarf rapeseed when NO3 was the nitrogen
source, p-value = 0.0301. Uppercase letters compares differences among N sources, and lowercase letters
compares differences between monoculture and mix. Flowering was quantified as flowers per stem. The
error bars represent ±1 standard error of the mean.
Discussion:
Tall rapeseed had greater biomass when grown with NO3, and greater flowering when
grown in monoculture. Perhaps the roots of tall rapeseed have a higher affinity for NO3 because
of the charge on the ion. The greater biomass of the NO3-supplied tall rapeseed individuals
could also be indicative of the use on NO3 within the plant. It is possible that NO3 is a more
usable form of nitrogen for the growth of shoots, while NH4 and glycine might have other
important physiological roles inside of the plant, leading to greater biomass of the NO3 treated
plants. Flowering was greater when tall rapeseed was in monoculture; this might be because
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interspecific competition is greater than intraspecific competition in this study system. When in
mixed pots, tall rapeseed has less energy to put towards reproduction, leading to lower quantities
of flowers, while if it is in monoculture it has sufficient energy to allocate to growth and
reproduction, causing higher levels of flowering in monoculture.
Dwarf rapeseed had greater biomass when grown in monoculture and greater flowering
when grown in a mix with NO3. It might be that dwarf rapeseed is more of a generalist, and that
is why it can grow equally well with any of the nitrogen sources used. For this reason, the N
source did not have a significant effect on the biomass. Biomass of dwarf rapeseed was greater
in monoculture than mixed because in mixed pots the tall rapeseed might have been outcompeting it for nutrients. It is interesting that the flowering of dwarf rapeseed was highest in
mixed culture with NO3; this might be due to a spurious result of some unknown bias. Because
biomass was greater in monoculture, it would have made sense for flowering to be higher also.
This study corroborates earlier studies that have found that plants are able to take up both
organic and inorganic nitrogen from the environment (Nasholm et al., 2000; Shimel and Bennett,
2004). This is important for plants that grow in soil where there is a limited supply of N. Instead
of depending on microbes to process the N into a form that they can use, they can compete more
directly with the microbes for usable nitrogen. Although NO3 was found to be a better source of
N for tall rapeseed with respect to biomass, this was only the case for dwarf rapeseed in mixed
pots. Furthermore, while the difference between biomass of tall rapeseed grown in NO3 and
those plants grown in NH4 and glycine is significant, it is not a large difference, demonstrating
that glycine and NH4 are also important and viable sources of N. Although much research has
focused on NO3, this study shows that it is not necessarily the only N source that can be taken up
from the soil. This might explain why plants can be successful in low productivity soil. Also,
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several species of plants can grow in the same microenvironment because they might take up soil
nutrients, like nitrogen, in different forms, reducing direct competition among plants.
There are some limitations of this study. Because each N source was tested individually,
it is unknown which form would be taken up the most if inorganic and organic N were
accessible. Perhaps the competition between dwarf and tall rapeseed would be different if there
were more than one form of nitrogen available for uptake. Future studies could explore this
aspect of nitrogen use. Another limitation is that this study was not done on different species,
but rather on two varieties of the same species. Interspecific competition would expected to be
different than what was observed in this study if two species had been used. Differences in
biomass and flowering might be greater with two species if they have varying competitive
abilities.
Appendix:
Figure 5. New paradigm of N cycling
Plants are able to compete with microbes more efficiently by taking up organic monomers as well as the
mineralized N sources that the microbes produce (Shimel and Bennett, 2004).
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Figure 6. Use of different forms of N based on productivity and N availability
Rapeseed is a Case A plant, an herb that is generally found in low productivity systems where N is
limited (Schimel and Bennett, 2004).
References:
Nasholm, T., K. Huss-Danell, and P. Hogberg. 2000. “Uptake of organic nitrogen in the field
by four agriculturally important plant species.” Ecology 81(4): 1155-1161.
Schimel, J.P. and J. Bennett. 2004. “Nitrogen mineralization: challenges of a changing
paradigm.” Ecology 85:591-602.
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