CSIRO PUBLISHING

Functional Plant Biology

http://dx.doi.org/10.1071/FP11240

Tomato response to legume cover crop and nitrogen:

differing enhancement patterns of fruit yield, photosynthesis
and gene expression

Tahira Fatima A,C, John R. Teasdale A, Jim Bunce B and Autar K. Mattoo A,D
A
Sustainable Agricultural Systems Laboratory, The Henry A. Wallace Beltsville Agricultural Research Center,
US Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705-2350, USA.
B
Crop Systems and Global Climate Change Laboratory, The Henry A. Wallace Beltsville Agricultural Research
Center, US Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705-2350, USA.
C
Present address: Department of Biology, The University of Western Ontario, London, ON N6A 4L9, Canada.
D
Corresponding author. Email: autar.mattoo@ars.usda.gov

Abstract. Excessive use of nitrogen (N) in crop production has impacted ecosystems by contaminating soil and water.
Management of N in agriculture is therefore of global concern. Sustainable agriculture systems that use leguminous cover
crops such as hairy vetch (Vicia villosa Roth) to fix N and enrich soil organic matter by fixing carbon provide an alternative
strategy. N signalling pathways were found associated with delayed leaf senescence and disease tolerance of hairy vetch-
grown tomatoes. To test whether N in hairy vetch is the only contributing factor leading to these phenotypes, we designed a
pot experiment in the field to analyse growth and gene expression in tomatoes, one set with soil overwintered without a cover
crop (bare soil) and the other with soil overwintered with a hairy vetch cover crop including the vetch residue on the soil
surface. Additionally, supplementary N fertiliser was also provided to aid distinguishing tomato responses to vetch from
those to inorganic N. Tomato fruit yield, plant biomass and photosynthesis were higher in plants grown in vetch than bare soil.
Tomato growth and photosynthesis metrics exhibited a parabolic response to inorganic N in bare soil, suggesting the potential
for N toxicity in pots with the highest N rate. Vetch-grown tomato plants mitigated these effects and maintained elevated
photosynthetic rates at high inorganic N levels. Vetch-grown plants also mitigated a decline in expression of several genes
regulating nitrogen and carbon metabolism and upregulated the defence-related gene, osmotin, relative to plants grown in
bare soil. Thus, some of the positive responses of tomatoes to a hairy vetch cover crop observed in the field seem mediated by
physiological cues other than the additional N provided by the vetch cover crop.

Additional keywords: biomass, nitrogen, plant growth, Solanum lycopersicum, sustainable agriculture.

Received 25 October 2011, accepted 20 January 2012, published online 14 March 2012

Introduction agricultural sustainability include organic farming with no

The burgeoning world population poses an increasing challenge
for food security. In the next 10–20 years, agriculturists will
need to double agricultural production using the existing,
cultivable land (Smaglik 2006). Conventional agriculture with
traditional breeding using non-renewable resources on the farm
has successfully met the production needs in many parts of the
world during the past century. However, this practice has also
led to the loss of topsoil, reduced soil fertility and excessive
use of synthetic pesticides (Smil 1997; Trewavas 2001) and
impacted world ecosystems because of heavy reliance on
synthetic fertiliser and fossil fuels (National Research Council
2010). Thus, the potential of alternative agriculture systems that
use sustainable inputs and enhance ecosystem services has
attracted considerable attention in recent years and provided a
research platform to further explore sustainable farming
systems (Mattoo and Teasdale 2010). Approaches to achieving
Journal compilation
CSIRO 2012
synthetic inputs, use of legume cover crops to replace or
decrease the use of synthetic fertiliser and modern genetic
tools that permit reductions in tillage and herbicides and
pesticides.
The production of fruits and vegetables is still heavily reliant
on the use of fertiliser and pesticides to optimise yield. During
the last decade or more, sustainable agriculture approaches have
included use of leguminous cover crops, such as hairy vetch
(Vicia villosa Roth), that fix atmospheric N, provides an on-farm
source of nitrogen (N) in the soil, fixes carbon that enriches soil
organic matter (Abdul-Baki and Teasdale 2007) and increases
microbial biomass (Buyer et al. 2010). Owing to these beneficial
properties, a hairy vetch cover crop can reduce soil erosion and
enrich fertility while increasing the yield, quality and profitability
of the produce (McVay et al. 1989; Rothrock et al. 1995; Abdul-
Baki et al. 1996; Creamer et al. 1996; Candole and Rothrock
www.publish.csiro.au/journals/fpb
B Functional Plant Biology
1997; Rice et al. 2001; Kumar et al. 2004; Neelam et al. 2008;
Mattoo and Teasdale 2010). Tomato plants grown in hairy vetch
(HV) residue showed longer life span and better tolerance
to diseases than those grown on black plastic (BP)
mulch (Teasdale and Abdul-Baki 1997; Kumar et al. 2004).
Further analysis of these plants showed higher expression of
genes involved in N assimilation, photosynthesis, carbon
mobilisation and defence response. Greenhouse experiments
conducted with HV residue taken from the field validated the
longer life span phenotype of tomato compared with bare soil
grown fresh market tomato (Kumar et al. 2005).
The source and nature of N fertiliser used to grow plants can
bring about differential gene expression responses, as shown in
wheat (Lu et al. 2005) and tomato (Mattoo and Handa 2008;
Neelam et al. 2008). HV residue is richer in N, so plants grown
in HV cover crop mulch in the field generally require half the
regular dose of a synthetic fertiliser (Abdul-Baki et al. 1997) and
N signalling pathways are associated with HV-grown tomatoes
(Teasdale and Abdul-Baki 1997; Mattoo and Abdul-Baki 2006)
suggesting that N in HV residue is the basis for the associated
phenotypes. We asked the question (Mattoo and Teasdale 2010):
is N in HV the only contributing factor leading to delayed leaf
senescence and disease tolerance of tomato plants grown in
legume cover crop? The basis of this question followed critical
analysis of the gene expression data (Kumar et al. 2004; Mattoo
and Abdul-Baki 2006) that did not completely match the patterns
seen in literature on N responsive genes. For instance, short-term
exposure to N upregulated the expression of senescence-
associated protein gene (SAG12) and nitrate transporter gene
(CHL1) whereas downregulating the expression of an antifungal
protein gene (osmotin) in Arabidopsis (Wang et al. 2000). In
contrast to these data, Kumar et al. (2004, 2005) found no change
in the expression of SAG12 or CHL1 transcripts while, indeed,
osmotin gene transcripts were actually elevated in HV-grown
tomatoes. There is a need to conduct full-season experiments
under field conditions to accurately assess plant responses from
the transcript to the agronomic level. To further test whether
the HV-mediated attributes are due solely to N, we designed an
experiment to analyse growth and gene expression in tomatoes
when grown in pots to permit more precise control over soil and
nitrogen conditions, but also when grown full season to maturity
under field environmental conditions. We established two
distinct sets of pots, one set with soil overwintered without a
cover crop (bare soil) and the other set with soil overwintered with
a hairy vetch cover crop (vetch amended) in the absence and
presence of supplemental N fertiliser. Plants were monitored for
photosynthesis, biomass, fruit yield, polyamine content and gene
expression of select set of genes. Overall, the data presented
suggest that hairy vetch cover crop mediates positive growth and
yield of tomatoes involving physiological cues other than just the
additional N.
Materials and methods
Hairy vetch growth
Raised beds were prepared on a Keyport fine sandy loam
(fine, mixed, semiactive, mesic Aquic Hapludults) field at the
North Farm of the USDA-ARS Beltsville Agricultural Research
Center, Beltsville, Maryland. Hairy vetch seeds were sown on
T. Fatima et al.
19 September 2006, using a Brillion seeder (Brillion Iron
Works, Brillion, WI) on one set of beds, while another set of
beds, hereafter designated as bare soil, were left unplanted. The
winter-annual hairy vetch cover crop established in the fall,
overwintered and developed abundant vegetative growth that
covered the bed by mid-spring. After vetch began flowering, the
aboveground biomass was mowed and removed from the field on
12 June 2007, air-dried in the hoop house for 3 days and used as a
mulch for the pot-grown tomato plants as described below.
Experimental design
Two soil treatments were established, one from the bare soil beds
and the other from the hairy vetch beds. Bare soil and vetch beds
were rotovated after vetch vegetation was removed and then
shovelled into designated pots (35 cm inner diameter, 35 cm tall)
with a designated soil to a 30 cm height (averaged 35.4 kg DW per
pot). Each pot was placed on a saucer with a 5 cm edge to facilitate
water retention. Two tomato (Solanum lycopersicum Mill. cv.
‘Sunbeam’) seedlings were transplanted into each pot on 13 June
2007. Each pot was thinned to one tomato plant after 3 weeks.
The pots with hairy vetch-grown soil received 400 g each of
air-dried hairy vetch residue pressed on top of the soil by hand
2 days after tomato seedlings had been transplanted to simulate
the no-tillage tomato production system with a hairy vetch cover
crop (Kumar et al. 2005). This corresponded to a dry weight
of 280 g and an N content of 7.92 g of vetch N added to each
pot, based on a vetch moisture content of ~30% and a vetch
tissue N concentration of 2.83%. This was the equivalent of
4000 kg ha–1 of dry vetch biomass with 113 kg N ha–1, assuming
that a typical field grown tomato plant grown for fresh market
occupies 0.7 m2.
Pots were arranged on landscape fabric that covered a level
section of the field in a randomised complete block design
with six replicates (see Fig. S1 available as Supplementary
Material to this paper). The average minimum and maximum
temperatures from transplanting to first harvest were 18 and
31C respectively.
Nitrogen and water application
Nitrogen in the form of ammonium nitrate was applied to plants
at the rates of 0, 3.5, 7 and 14 g N per pot, equivalent to rates of
0, 50, 100 and 200 kg N ha–1 in the field. This total N rate was
distributed across 10 equal weekly doses starting 7 days after
transplanting. A soluble form of this fertiliser was applied in
solution as part of the daily watering regime. Pots were watered
daily according to a protocol designed to maintain soil moisture
at approximately field capacity and avoid either excess or
deficient conditions. In addition saucers beneath each pot were
filled with water to maintain a source of water from capillary
movement as the soil dried each day. There were highly variable
water requirements resulting from a range of growth rates
induced by variable N rates and from the presence or absence
of a vetch mulch, so a soil moisture sensor (Field Scout TDR
300, Spectrum Technologies Inc., Plainfield, IL, USA) was used
to guide watering of each pot each day. A threshold soil
volumetric water content of 20% in addition to the degree of
depletion of saucer water were used to determine daily watering
requirements and maintain each pot at a similar water status.
Tomato response to legume cover crop and nitrogen
Analysis of soil mineral content
Soil samples from bare and hairy vetch field beds, collected at
the time pots were filled, were analysed by A&L Laboratories
(Richmond, VA, USA) for content of macronutrients. The hairy
vetch residue used for the soil surface of the vetch pots was
analysed by A&L Laboratories for N concentration.
Photosynthesis and stomatal conductance measurements
Single leaf CO2 and H2O vapour exchange measurements were
made on clear days using a CIRAS-1 portable photosynthesis
system (PP Systems, Amesbury, MA) under ambient mid-day
conditions of light, temperature, RH and CO2 concentration,
using a broad-leaf cuvette. Measurements were made on 48,
57 and 72 days after transplanting (DAT) (31 July, 9 August
and 24 August). Leaf temperatures ranged from 26 to 30C across
all measurement dates. PPFD exceeded 1200 mmol m–2 s–1 for
all measurements and the CO2 concentration external to the
leaves was controlled at 370 mmol mol–1. Leaves selected for
measurement were recently fully expanded upper canopy leaves
fully exposed to light and were found to have the highest rates
of any leaves on the plants. On each date, gas exchange was
measured on one leaf of each plant in all replicates. The order of
measurements of treatment within hairy vetch and bare soil was
random. Measurements were completed in ~2 h on each date.
Polyamine analysis
Leaves harvested on 75 DAT (27 August) were processed and
analysed for their content of polyamines, (putrescine (Put),
spermidine (Spd) and spermine (Spm) levels) as previously
described (Minocha et al. 1990; Mehta et al. 2002).
Fruit yield, biomass and leaf senescence measurements
Fruit yield was determined by harvesting fruit at breaker or
turning stage from all six replicates in each treatment.
Harvesting of fruit began on 77 DAT and continued
approximately weekly until 112 DAT (29 August–3 October).
Leaf senescence was assessed on 34, 76, 83 and 91 DAT
(17 July, 28 August, 4 September and 12 September) by
estimating the percentage of senescent leaves on each plant.
When plants for a specific treatment became highly senescent
and fruit production was declining, a final fruit harvest was
conducted and vegetative biomass was collected. Accordingly,
biomass harvest of the bare soil/0 N treatment was conducted on
29 August, the bare soil/50 N treatment on 11 September, the bare
soil/100 and 200 N treatments on 19 September and the vetch
treatments on 3 October. Both FW and DW of fruit collected at
each harvest date were determined as well as cumulative fruit
weights. After plant harvest, the plant was separated into fruit,
aboveground vegetative matter and belowground root matter and
then dried at 80C until constant weight.
Quantitative PCR analysis
Leaves were harvested from plants receiving 0, 100, or
200 kg N ha–1 on 75 and 89 DAT (27 August and 10
September) and freeze dried. Total RNA was isolated from
lyophilised samples from two biological replicates using
the RNeasy Plant Mini Kit (Qiagen, Frederick, MD, USA).
For all tissues a single RNA extraction was performed. RNA
Functional Plant Biology C
concentration and quality were determined using a
spectrophotometer and native agarose gel electrophoresis
respectively. First strand cDNA synthesis was performed
using the SuperScript cDNA Synthesis Kit (Invitrogen, Grand
Island, NY, USA) following the manufacturer’s instructions,
in a final volume of 20 mL. The final cDNA products were
diluted 10-fold before use in real-time RT. Quantitative
RT–PCR was performed with gene-specific primers (see
Table S1 available as Supplementary Material to this paper),
which were designed using Primer3 (http://primer3.sourceforge.
net) software. The RT–PCR conditions were the same as before
(Mattoo et al. 2006). Actin gene from tomato was used as
reference for normalisation. Transcripts of 10 genes listed in
Table S1were quantified in leaf samples from plants grown in
bare and HV-amended conditions by the comparative CT method
using the 2–DDCT formula as described by Livak and Schmittgen
(2001) and Mattoo et al. (2006).
Statistical analyses
Analysis of variance was performed on all data collected using
the mixed model procedure of SAS ver. 9.1 (SAS Institute, Cary,
NC). Soil (bare or vetch) and nitrogen rate were treated as a class
variables. If this analysis showed significant effects (P < 0.05)
and there was visual evidence of a linear or quadratic response
across N rates, then an analysis of covariance was performed
with soil as a class variable and N rate as a regression variable.
Analysis of variance was performed on target gene expression
computed as 2–DDCT. Sample date for gene expression was treated
as a random variable since sample dates were only 2 weeks
apart. Variance was partitioned to adjust for the heterogeneity
of variance encountered in gene expression data. In addition,
threshold cycle (CT) values for the reference gene, actin, were
subjected to an analysis of variance to ensure that expression of
this gene was unaffected by experimental treatments as
recommended (Livak and Schmittgen 2001). This analysis
showed no significant (P < 0.05) treatment influences on actin
expression, except that associated with glutamine synthetase
(GS2).
Results
Mineral content of bare soil and hairy vetch-grown soil
The initial mineral content of soils without a cover crop (bare) or
with a hairy vetch cover crop (vetch) was determined. The content
of phosphorus, magnesium, calcium and ammonium-N were
similar in both bare and vetch soils (Table 1). However, the
content of potassium and nitrate-N was higher in vetch than bare
soil. Although there was greater potassium in vetch soil, both soils
were rated as having ‘high’ levels for tomatoes by the soil testing
laboratory. The 33 mg kg–1 difference in nitrate-N between soils
was equivalent to 1.17 g N pot–1 or an estimated 16.7 kg N ha–1
advantage for the vetch soil. The N content in the vetch residue
added to each pot was 7.92 g N pot–1, however, since only about
half of vetch N content is usually available in a given year
(Rosecrance et al. 2000; Seo et al. 2006), only ~3.96 g N pot–1
would have been available to the tomato plants in this experiment.
Thus, the vetch treatment would be expected to contribute an
estimated 1.17 g N pot–1 from soil nitrate and 3.96 g N pot–1 from
D Functional Plant Biology T. Fatima et al.

Table 1. Mineral content of soils without vetch (bare) and with a hairy vetch cover crop that were utilised in this experiment
Analytical method: Mehlich III. Values of P, K, Mg and Ca for both soils were rated as ‘very high’, ‘high’, ‘medium’ and ‘medium’, respectively, by A&L
Laboratories. Nitrate-N was rated as ‘high’ for bare soil and ‘very high’ for vetch soil. Mean is shown with the standard deviation in parentheses

Soil P (mg kg–1) K (mg kg–1) Mg (mg kg–1) Ca (mg kg–1) Nitrate-N (mg kg–1) Ammonia-N (mg kg–1)
Bare 378 (06) 125 (07) 102 (3) 820 (27) 23 (1) 11 (8)
Vetch 371 (15) 173 (16) 98 (6) 783 (42) 56 (5) 11 (7)

vetch residue totalling 5.13 g N pot–1 (equivalent to 73 kg N ha–1)
more nitrogen than the bare soil treatment.
Fruit yield, biomass and N uptake
Analysis of covariance showed a significant difference between
vetch and bare soil and a significant quadratic response to N rate,
but no significant interaction between soil and N rate (Fig. 1a).
Total average tomato fruit yield in the absence of supplemental N
was 213 g plant–1 and 1108 g plant–1 for bare soil and soil plus
hairy vetch respectively. Maximum fruit yield was obtained at
100 kg N ha–1 in both soil types, which declined in both cases at
200 kg N ha–1 (Fig. 1a). Analysis of covariance of biomass and
N uptake data showed a similar pattern of significant soil
effects, significant quadratic response to N rate and lack of soil
by N rate interactions. Biomass DW variables and total N uptake
were higher in vetch-amended than bare soil pots, but tended
to increase quadratically to N rate with often little difference
between the 100 and 200 kg N ha–1 rates (Table 2). However,
N utilisation efficiency was similar between the two soils and
declined linearly with N rate (Table 2).

Photosynthetic rates of leaves at fruit set and fruit harvest

(a) Photosynthesis measurements were recorded on 48, 57 and 72
DAT on fully expanded leaves. Fruit setting on plants occurred
around 58 DAT while 73 DAT was just before the beginning
of ripe fruit harvest. Consistent with the data on fruit yield and
total biomass, tomato leaf photosynthesis was higher in plants
grown on vetch than bare soil at almost all N rates at the three
measurement dates (Fig. 2). Photosynthesis trended slightly
upward with increasing N rate in vetch soil (Fig. 2a) but in
bare soil grown plants it increased to a peak at 50–100 kg N ha–1,
declining at the 200 kg N ha–1 rate (Fig. 2b). Compared with
photosynthetic rates before and at fruit setting, leaf
Fruit yield (kg plant–1)

photosynthesis declined at all N rates in bare soil when the

fruit were at an advanced stage of ripeness; however, the
decline was more pronounced in plants grown in bare than
(b) vetch soil.

Vetch-amended soil grown plants have altered leaf

Nitrogen (kg ha–1)
Fig. 1. Tomato fresh fruit yield when grown in pots with bare soil or soil
and residue from a hairy vetch cover crop at selected fertiliser nitrogen rates.
(a) Measured tomato fruit yield showing mean points with standard error
bars and fitted polynomial models, Y = 213 + 36.9N – 0.153N2 for bare soil
and Y = 1108 + 36.9N – 0.153N2 for vetch, where Y is fruit yield and N is
nitrogen. (b) Actual polynomial modelled yield for tomato grown in bare
soil (from a) and hypothetical polynomial modelled yield for tomato
grown in vetch soil if the only influence of vetch was to add an additional
73 kg N ha–1 to the pots otherwise defined by the bare soil model,
Y = 213 + 36.9(N + 73) – 0.153(N + 73)2.
putrescine and spermine patterns
Biogenic amines such as diamine putrescine (Put), triamine
spermidine (Spd) and tetraamine spermine (Spm) are organic
N signalling molecules that are known to respond to N status
(Bauer et al. 2004) and environmental changes (Alcázar et al.
2006). Therefore, their contents were determined in leaves of
tomato plants grown on bare and vetch-amended soils on 75 DAT
when fruit were at the advanced stage of ripeness. Put was the
most abundant of polyamines present in leaves; its content was
substantially higher in leaves of plants grown in bare soil at 100
and 200 kg N ha–1 than in all other treatments (Table 3). Leaf
Spd content was no different in bare and vetch-amended soils
and remained unaffected by the treatments. However, Spm was
higher on average in plants grown in vetch (12.6 nmol g–1)
than bare soil (4.9 nmol g–1). High photosynthetic levels in
vetch plants at 100 and 200 kg N ha–1 on 24 August (72 DAT)
compared with low photosynthetic rates at these same N rates
in bare soil (Fig. 2) inversely mirrored the pattern of Put data
and provides evidence of the different response patterns of vetch
and bare soil grown plants to high N rates.
Tomato response to legume cover crop and nitrogen Functional Plant Biology E

Table 2. Tomato plant dry weight and nitrogen uptake at harvest and nitrogen utilisation efficiency for plants grown in pots with
bare soil or soil and residue from a hairy vetch cover crop at selected fertiliser nitrogen rates
N utilisation efficiency was computed as the dry fruit yield per total plant N. Standard error values are shown in parentheses. Analysis of
covariance results where soil is a class variable and N is a regression variable is shown at the bottom of the table. Significant effects are
indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant

Soil Nitrogen rate Vegetative DW Fruit DW Total DW Total N uptake N utilisation

(kg ha–1) (g plant–1) (g plant–1) (g plant–1) (g plant–1) efficiency
(g g–1)
Bare 0 36 (03) 36 (12) 72 (12) 0.97 (0.19) 31.2 (7.1)
50 75 (06) 64 (03) 139 (07) 2.69 (0.13) 23.9 (1.0)
100 101 (09) 62 (03) 163 (08) 4.30 (0.25) 14.6 (1.1)
200 94 (04) 72 (09) 166 (10) 5.60 (0.29) 12.8 (1.2)
Vetch 0 64 (05) 41 (03) 105 (03) 1.66 (0.06) 25.1 (2.1)
50 86 (03) 82 (08) 167 (09) 3.06 (0.41) 27.8 (3.2)
100 121 (11) 112 (15) 233 (25) 5.86 (0.81) 19.4 (1.1)
200 117 (09) 105 (13) 222 (22) 5.65 (0.44) 18.3 (1.7)
Effect F-test
Soil – *** *** *** * ns
N linear – *** *** *** *** ***
N quadratic – *** ** *** *** ns
N  soil – ns ns ns ns ns
N  N  soil – ns ns ns ns ns

Table 3. Tomato leaf polyamine content at the onset of fruit harvest for
(a) plants grown with bare or hairy vetch soil at selected fertiliser N rates
Standard error values are shown in parentheses (no s.e. is shown for
200 N kg ha–1 for vetch because lost samples resulted in only one value for
this treatment). Analysis of variance results are shown at the bottom of the
table. Significant effects are indicated: *, P < 0.05; **, P < 0.01; ***,
P < 0.001; ns, not significant

Soil Nitrogen rate Putrescine Spermidine Spermine

48 DAT
(kg ha–1) (nmol g–1) (nmol g–1) (nmol g–1)
57 DAT
Bare 0 43 (10) 26 (4) 7.9 (2.0)
PN (µmol CO2 m–2 s–1)

72 DAT 50 103 (27) 32 (4) 5.8 (0.7)

100 460 (66) 42 (1) 2.7 (3.1)
200 490 (36) 28 (5) 3.3 (3.1)
Vetch 0 87 (25) 47 (9) 16.2 (2.8)
50 35 (02) 29 (2) 5.2 (1.5)
(b)
100 68 (17) 36 (5) 8.8 (1.5)
200 126 38 20.2
Effect F-test
Soil *** ns **
N *** ns *
Soil  N *** ns ns

Patterns of N responsive, N : C signalling indicator

Nitrogen (kg ha–1)
Fig. 2. Mid-morning net photosynthetic rate (PN) of tomato plants grown
in vetch (a) or bare (b) soil in response to nitrogen rate on 48 days after
transplanting (DAT) and 57 DAT (fruit setting) and 72 DAT (shortly before
the beginning of ripe fruit harvest).
and defence-related gene transcripts
A battery of genes were selected to determine their expression in
the leaves of tomato plants grown on bare soil and soil-amended
with hairy vetch and in response to supplementary N. The
selection was made on the basis of their functional activity in
the cell; N and NH4 uptake/assimilating genes; nitrate reductase
(NR), nitrite reductase (NiR), aspartate aminotransferase
(ASAT) and glutamine synthetase (GS2); organic acid and
phospo-pentose pathway and N : C indicator genes: cytosolic
F Functional Plant Biology
isocitrate dehydrogenase (ICDH), phospho-enolpyruvate
carboxylase (PEPC) and glucose-6-phosphate dehydrogenase
(G6PDH); secondary metabolite pathway gene phenylalanine
lyase (PAL); defence-related genes: osmotin (OSM) and
cytokinin receptor kinase (CKR). Leaf tissue from plants was
analysed for transcript levels using quantitative polymerase chain
reaction (Q-PCR) as described in ‘Materials and methods’.
ANOVA was applied to the gene expression datasets to find
significant differences in the leaf gene transcripts between the
two soil types in the absence and presence of supplemental N
(Table 4). Because there was considerable modulation in this
data, only the most consistent effects that were significant at
P < 0.01 are presented graphically and discussed in detail. Two
genes, NR and OSM, were upregulated in vetch versus bare soil.
Both these genes had a significant main effect for soil with
higher gene expression in vetch than bare soil at all nitrogen
rates (Fig. 3a, b; Fig. S2). NR expression was increased in vetch
(average = 1.29 across all N rates), whereas it was decreased
in bare soil (average = 0.54). OSM expression was generally
increased by N treatments, but the increase was greater in
vetch than bare soil at all N rates (average expression was
9.8 and 3.2 in vetch and bare soils respectively). OSM
expression tended to peak at 100 kg N ha–1 in bare soil, but
was synergistically increased by the N supplementation of
200 kg N ha–1 plus vetch-soil.
PEPC and GS2 had significant soil by N interactions
(Table 4) whereby expression was higher in bare soil than in
vetch soil only under selected N conditions. PEPC transcripts
were higher in leaves from plants grown on bare soil treated
with 100 kg N ha–1 but this effect of N was not evident at 0 and
200 kg N ha–1 (Fig. 3c; Fig S3). The highest GS2 transcripts
were found in leaves of plants grown in bare soil in the
absence of added N accounting for significant ANOVA effects
(Table 4). However, there was a significant soil by sampling date
interaction (P = 0.04) for the actin reference gene associated
with GS2 and this was driven by higher actin CT values in the
bare soil 0 and 100 N treatments at the first sampling date (25.3
and 23.1 respectively) than in all other treatments (average = 1.0).
This differential would tend to inflate the DDCT values of all other
treatments, which would then deflate the 2–DDCT values. Thus, the
higher GS2 bare soil values in the absence of N are probably a
consequence of treatment effects on the actin reference gene
rather than on the target GS2 gene.
NiR, CKR, PAL and G6PDH showed some treatment effects
at P < 0.1 (Table 4), but these were driven by increases in gene
activity in vetch soil at only one N rate at one sampling date and
were not consistently expressed (Figs S4, S5). Overall gene
Table 4. Analysis of variance of gene responses to soil (bare or hairy vetch) and nitrogen rate
NR, nitrate reductase; NIR, nitrite reductase; CKR, cytokinin receptor kinase; PEPC, phosphoenolpyruvate carboxylase; PAL, phenylalanine ammonia lyase;
GS2, glutamine synthetase; OSM, osmotin; ASAT, aspartate aminotransferase; G6P, glucose-6-phosphate dehydrogenase; ICDH, cytosolic isocitrate
dehydrogenase. Significance is indicated: **, P < 0.01
Effect
NR NIR CKR PEPC
Soil 0.000** 0.093 0.054 0.000**
N 0.002** 0.034 0.300 0.000**
Soil  N 0.369 0.077 0.067 0.000**
T. Fatima et al.
expression was higher for NiR (mean = 1.5 across all soil and
N treatments), CKR (mean = 2.1) and ICDH (mean = 1.3),
whereas gene expression was lowered for PAL (mean = 0.56),
ASAT (mean = 0.73) and G6PDH (mean = 0.68).
Discussion
Tomato exhibits several phenotypic responses to hairy vetch
cover crop in the field resulting in higher leaf area duration
(Teasdale and Abdul-Baki 1997) and higher fruit yield
(Abdul-Baki et al. 1996) compared with a conventionally-
grown crop. These responses require full-season evaluation in
the field to demonstrate. Physiological behaviour of plants
grown in short-term growth chamber experiments often does
not parallel that of field-grown plants (Burkey and Wells 1991;
Conesa et al. 2007). In this paper, we provide an experimental
approach for evaluating genotypic responses of tomatoes grown
full-season in a field setting, but with more precise controls
over nitrogen and soil conditions in large pots to overcome
the heterogeneity found in field soils at the level of the
individual plant. We demonstrate here that longer duration of
photosynthesis, higher fruit yield and higher vegetative growth of
tomato plants grown in hairy vetch-amended soil compared with
plants grown in bare soil is associated with differential changes in
biogenic amine content and select gene expression. However,
such long-term field experimentation with agronomic treatments
may require additional evaluation of reference gene response
to treatments during qRT–PCR analysis to ensure their
unresponsiveness to treatments relative to responses of target
genes (Livak and Schmittgen 2001). We found a significant
treatment effect on our actin reference gene in only one of 10
genes. Additional research is needed to identify reference genes
that are relatively unaffected by full-season agronomic treatments
under field conditions.
A quadratic response to N rate was apparent with maximum
fresh fruit yield (Fig. 1a) and dry biomass (Table 2) occurring at
~100 kg N ha–1 followed by a slight decline at 200 kg N ha–1.
There was no interaction between soil and nitrogen for fresh fruit
yield and all biomass variables, implying that the same quadratic
model defined the response of both vetch and bare soil to N, but
with a higher intercept for vetch than bare soil (Fig. 1a). If the only
effect of vetch on tomato growth was the addition of 73 kg N ha–1
(the estimated differential between vetch and bare soil treatments)
then the maximum fruit yield should not change – instead, the
curve should just shift towards the origin (Fig. 1b). However, the
fact that actual results showed higher yield and biomass in vetch
than bare soil at all N levels, a response that is distinctly different
Probability > F
PAL GS2 OSM ASAT G6PDH ICDH
0.037 0.041 0.000** 0.414 0.285 0.952
0.193 0.000** 0.000** 0.184 0.041 0.611
0.374 0.001** 0.005** 0.735 0.498 0.797
Tomato response to legume cover crop and nitrogen
(a)
NR
(b)
Relative transcript level
OSM
(c)
PEPC
Nitrogen rate (kg ha–1)
Fig. 3. Relative response of (a) nitrate reductase (NR), (b) osmotin (OSM)
and phosphoenolpyruvate carboxylase (PEPC) transcripts in leaves of tomato
plants grown on bare soil (bare) versus vetch-amended soil (HV) in the
absence and presence of the two indicated doses of supplemental inorganic
N. Means are pooled over four values derived from two replications each of
two harvest dates at 75 and 89 days after transplanting (DAT). Error bars
represent the s.e. A response of 1.0 corresponds to the transcript level for
the bare soil treatment without supplemental N at 75 DAT. NR, accession #
AW218786.1; OSM, accession # AW218786.1; PEPC, accession #
AJ313434.
from the hypothetical result shown in Fig. 1b, suggests that
tomato response to vetch was defined, in part, by factors
different than nitrogen.
The quadratic response of many variables to N suggests that
slightly inhibitory levels of N were present in pots at the highest
N rate. Tomato fruit yield, plant biomass and late-season
photosynthesis showed this tendency. Excess levels of N could
have led to nitrate/ammonia poisoning, tomato is known to be
sensitive to such toxicity (Feng and Barker 1993; Britto and
Kronzucker 2002) in various degrees, particularly in the late
season bare soil pots where photosynthesis was severely
depressed at the 100 and 200 kg N ha–1 rates (Fig. 2b). The
high levels of putrescine accumulated in the leaves of these
same bare soil treatments sampled three days after the late
Functional Plant Biology G
photosynthesis measurements (Table 3), suggests the presence
of potentially toxic levels of N in these tissues at that time (Feng
and Barker 1993; Wargo et al. 2002; Houdusse et al. 2005). It is
notable that the hairy vetch treatment mitigated this effect on
tomato photosynthesis and putrescine accumulation at high N
rates and did not exhibit any greater yield decline at the high N rate
than that in bare soil. If the primary effect of hairy vetch was to
provide supplementary N, then a greater decline in yield and
photosynthesis would have been expected at the high N rates than
that observed in bare soil. Since the opposite was observed, this is
further evidence that hairy vetch influenced tomato by processes
other than the simple addition of N.
Most revealing are the data on gene transcript levels. There
was little systematic effect of N rate alone on the expression of
selected genes in this experiment. Clearly, the response of genes
studied here to vetch-grown versus bare soil exhibited a more
complex pattern than what would be expected from plants simply
supplemented with N associated with the vetch cover crop. Thus,
downregulation of NR in bare soil was prevented in vetch-grown
plants whereas greater upregulation of osmotin was observed in
vetch than bare soil treated plants across all N and sampling
conditions. Some of these data differ from studies in the literature
involving short-term exposure of plants or cell cultures to
supplemental N under controlled conditions (Wang et al.
2000; Mattoo and Teasdale 2010). These data demonstrate that
the vetch cover crop-mediated influence on physiological
processes of tomato occurred in a manner other than through
simply providing additional nitrogen to the system. The fact
that hairy vetch residue was richer by 59% over bare soil in
nitrate-N (Table 1) would likely contribute to regulation of
N-responsive genes – NR, osmotin and GS2 (Kumar et al.
2004). Osmotin transcript abundance is synergistically
stimulated by N (200 kg N ha–1) supplementation of vetch-
amended soil. N stimulation of PEPC transcripts in bare
soil-grown tomato plants observed here was previously seen in
N-supplemented detached leaf segments of maize (Sugiharto
and Sugiyama 1992), transgenic tomato fruits that accumulate
organic N in the form of Spd and Spm (Mattoo et al. 2006)
and HV-grown transgenic tomatoes (Neelam et al. 2008). The
lesser PEPC response of plants in vetch than in bare soil to
N further indicates that, in addition to responses to exogenous N,
specific HV  N interactions differentially regulate certain gene
responses.
Another significant component, other than nitrate-N, present
in the vetch residue is potassium, 28% higher content in vetch
than bare soil. As a major osmoticum and a catalytic ligand for
several enzymatic reactions, potassium is important for
photosynthesis and growth of plants (White and Karley 2010).
Potassium deficiency causes early senescence likely via
induction of genes involved in ethylene biosynthesis and
ethylene signalling (Shin and Schachtman 2004; Jung et al.
2009). Thus, enrichment of vetch residue with potassium
could have contributed to stimulating growth and preventing
early senescence, being synchronous with other positive aspects
of vetch. This is particularly relevant in the context of previous
reports on potassium effects on horticultural crops. For instance,
application of potassium significantly increased total yield,
colour intensity and mean fruit weight of a processing variety
of tomato (Karkas 1992; Hartz et al. 2005; Akhtar et al. 2010);
H Functional Plant Biology
quickened germination, fast emergence rates and increased plant
leaf area (Alvardo et al. 1987); and improved nutritional quality of
tomato fruit by producing more lycopene (Ozbun et al. 1974).
Intensive potassium nutrition was recommended for high total
yields, quality parameters, marketable yield, brix, colour, size and
lycopene of processed tomato (Oded and Uzi 2003). Likewise, a
22–40% increase in yield and improved quality was found in
peppers, cucumbers and squash fertigated with potassium nitrate
(Johnstone et al. 2001). However, in our experiment, potassium
levels were not deficient in the bare soil treatment, but both soils
were categorised as having ‘high’ levels by the testing laboratory.
Therefore, greater potassium in vetch soils may have contributed
to the enhanced tomato growth response to vetch, but was
probably not the driver of this difference.
The responses of selected agronomic and physiological
variables to soil treatment with hairy vetch and to inorganic N
confirm that hairy vetch influences tomato by processes other than
the simple addition of N. More detailed research is required to
catalogue a more comprehensive list of transcriptional changes
in response to hairy vetch and the growth stages at which
they occur. Identification of the specific signalling molecular
triggers and receptors would provide insight into the ecological
processes leading from degradation of this legume cover crop
to altered responses in the tomato plant. Knowledge of these
processes could permit development of more precise cover crop
management practices in sustainable production systems.
Acknowledgements
We thank Ruth Mangum and Peter Ewashkow for help in setting up and
harvesting this experiment. Mention of trade names or commercial products in
this publication is solely for the purpose of providing specific information and
does not imply recommendation or endorsement by the USA Department of
Agriculture.
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