Novel and conserved functions of S-nitrosoglutathione reductase (GSNOR) in tomato

Abstract

Nitric oxide (NO) is emerging as a key signalling molecule in plants. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by depleting S-nitrosoglutathione (GSNO), the major cellular NO donor. Here we show that depletion of GSNOR function impacts tomato (Solanum lycopersicum. L) fruit development. Thus, reduction of GSNOR expression through RNA interference (RNAi), modulated both fruit formation and yield, establishing a novel function for GSNOR. Further, depletion of S. lycopersicum GSNOR (SlGSNOR) additionally impacted a number of other developmental processes, including seed development, which also has not been previously linked with GSNOR activity. In contrast to Arabidopsis, depletion of GSNOR function did not influence root development. Further, reduction of GSNOR transcript abundance compromised plant immunity. Surprisingly, this was in contrast to previous data in Arabidopsis that reported reducing Arabidopsis thaliana GSNOR (AtGSNOR) expression by antisense technology increased disease resistance. We also show increased SlGSNOR expression enhanced pathogen protection, uncovering a potential strategy to enhance disease resistance in crop plants. Collectively, our findings reveal at the genetic level, some but not all GSNOR activities are conserved out with the Arabidopsis reference system. Thus, manipulating the extent of GSNOR expression may control important agricultural traits in tomato and possibly other crop plants.

Full text

Journal of Experimental Botany, Vol. 70, No. 18 pp. 4877–4886, 2019
doi:10.1093/jxb/erz234 Advance Access Publication 14 May, 2019
This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details)
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RESEARCH PAPER
Novel and conserved functions of S-nitrosoglutathione
reductase intomato
Adil Hussain1,5, Byung-Wook Yun2, JiHyun Kim3, KapugantiJagadis Gupta4,, Nam-In Hyung3 and
GaryJ. Loake5,*,
1 Department of Agriculture, Abdul Wali Khan University Mardan, Khyber-Pakhtunkhwa, Pakistan
2 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Republic of Korea
3 Department of Plant and Food Sciences, Sangmyung University, Cheonan, Republic of Korea
4 National Institute of Plant Genome Research, Aruna Asaf Ali Marg, 110067, Delhi, India
5 Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, King’s Buildings, Mayfield Road,
Edinburgh EH9 3BF, UK
* Correspondence: gloake@ed.ac.uk
Received 15 March 2019; Editorial decision 29 April 2019; Accepted 29 April 2019
Editor: Stanislav Kopriva, University of Cologne, Germany
Abstract
Nitric oxide (NO) is emerging as a key signalling molecule in plants. The chief mechanism for the transfer of NO
bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an
S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cel-
lular S-nitrosylation, by depleting S-nitrosoglutathione (GSNO), the major cellular NO donor. Here we show that de-
pletion of GSNOR function impacts tomato (Solanum lycopersicum. L) fruit development. Thus, reduction of GSNOR
expression through RNAi modulated both fruit formation and yield, establishing a novel function for GSNOR. Further,
depletion of S. lycopersicum GSNOR (SlGSNOR) additionally impacted a number of other developmental processes,
including seed development, which also has not been previously linked with GSNOR activity. In contrast to Arabidopsis,
depletion of GSNOR function did not influence root development. Further, reduction of GSNOR transcript abundance
compromised plant immunity. Surprisingly, this was in contrast to previous data in Arabidopsis that reported that re-
ducing Arabidopsis thaliana GSNOR (AtGSNOR) expression by antisense technology increased disease resistance.
We also show that increased SlGSNOR expression enhanced pathogen protection, uncovering a potential strategy to
enhance disease resistance in crop plants. Collectively, our findings reveal, at the genetic level, that some but not all
GSNOR activities are conserved outside the Arabidopsis reference system. Thus, manipulating the extent of GSNOR
expression may control important agricultural traits in tomato and possibly other crop plants.
Keywords: Climacteric fruit, fruit development, GSNOR, MicroTom, nitric oxide, NO, S-nitrosation, S-nitrosylation, tomato,
tomato fruit.
Introduction
Nitric oxide (NO) underpins a plethora of cellular processes
integral to the biology of plants. The chief mechanism for the
transfer of NO bioactivity is thought to be S-nitrosylation, the
addition of an NO moiety to a peptide or protein cysteine thiol
to form an S-nitrosothiol (SNO) (Spadaro etal., 2010). This
redox-based post-translational modication controls a number
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4878 | Hussain etal.
of key activities related to growth, development, and environ-
mental interactions, including immune function. Typically, pro-
tein SNOs can be denitrosylated by the antioxidant tripeptide,
glutathione (GSH), resulting in the reconstitution of the pro-
tein Cys thiol and formation of S-nitrosogluthionine (GSNO)
(Airaki etal., 2011), which functions as a natural NO donor
and consequently a reservoir for NO bioactivity (Feechan
etal., 2005).
S-Nitrosoglutathione reductase (GSNOR), rst identied
in bacteria (Liu etal., 2001), is thought to be the major deter-
minant in the control of total cellular SNO levels in Arabidopsis
(Feechan etal., 2005; Lee etal., 2008; Leterrier etal., 2011). The
enzyme has a high anity for GSNO (Liu etal., 2001; Achkor
etal., 2003). Loss-of-function mutations in AtGSNOR1 com-
promise multiple modes of plant disease resistance, while
overexpression of this gene conveys increased disease resistance.
Further, AtGSNOR1 has been shown to regulate both salicylic
acid (SA) biosynthesis and associated signalling (Feechan etal.,
2005; Rustérucci etal., 2007; Tada etal., 2008).
In the context of SA signalling (Loake and Grant, 2007; Fu
and Dong, 2013) , S-nitrosylation of the A.thaliana SA-binding
protein 3 (AtSABP3) at Cys280 suppresses its binding to both
the immune activator, SA, and the carbonic anhydrase activity
of this protein, negatively regulating disease resistance (Y.J.
Wang et al., 2009). Further, NO via GSNO has been shown
to protect the TGA1 transcriptional regulator from oxygen-
mediated modications and enhance the DNA binding activity
of this protein to its cognate cis-element in the presence of
the transcriptional co-activator, NPR1. In addition, the trans-
location of NPR1 into the nucleus may be promoted by NO
(Lindermayr etal., 2010). In contrast, GSNO accumulation in
atgsnor1-3 plants has been reported to inhibit the translocation
of NPR1 from the cytoplasm to the nucleus, thereby curbing
SA signalling and associated plant immunity (Tada etal., 2008;
Yun et al., 2016). NO is also proposed to play a central role
in signalling activated by the fungal elicitor, cryptogein (Kulik
etal., 2015), and is required for disease resistance against Botrytis
cinerea triggered by oligogalacturonides (Rasul etal., 2012).
Both NO and reactive oxygen intermediates (ROIs) have
been implicated in the programmed cell death of pathogen-
challenged cells through the hypersensitive response (HR;
Delledonne et al., 1998, 2001; Torres et al., 2002). Aloss-of-
function allele of atgsnor1 [paraquat resistance 1-2 (par1-2)] con-
veyed protection against cell death mediated by the herbicide,
paraquat (Chen et al., 2009). Further, NO has been shown
to regulate the production of ROIs by the S-nitrosylation
of NADPH oxidase (AtRBOHD) at Cys890, reducing ROI
production at later stages of the plant defence, curbing devel-
opment of the HR (Yun et al., 2011). Interestingly, oxidative
post-translational modication of GSNOR inhibited the ac-
tivity of this enzyme, suggesting an additional mechanism of
direct crosstalk between ROI and NO signalling (Frungillo
etal., 2014; Guerra etal., 2016; Kovacs etal., 2016; Lindermayr,
2018).
AtGSNOR1 has also been shown to control some key
aspects of plant development (Lee etal., 2008; Leterrier etal.,
2011; Kwon etal., 2012). For example, atgsnor1-3 mutants show
loss of apical dominance, a subtle change in leaf shape, and
increased sensitivity to auxin (Kwon et al., 2012). This line is
also reduced in fertility, principally due to very short stamens
which do not function as eective self-pollinators (Kwon etal.,
2012; Xu etal., 2013). AtGSNOR1 has also been implicated in
responses to abiotic stress (Corpas etal., 2011; Fancy etal., 2017;
Begara-Morales etal., 2018). Missense alleles of hot5/atgsnor/
par2 cannot acclimate to heat as do dark-grown seedlings, but
grow normally and can heat-acclimate in the light. In contrast,
null alleles cannot heat-acclimate like light-grown plants (Lee
etal., 2008). Thus, AtGSNOR is required for heat acclimation.
In sunower seedlings exposed to high temperature (38°C for
4h), GSNOR activity, protein levels, and transcript abundance
have been found to be reduced in hypocotyls, with the sim-
ultaneous accumulation of SNOs (Leterrier et al., 2011). The
consequence was a rise in protein tyrosine nitration, which
is considered a marker of nitrosative stress. Collectively, these
ndings imply that AtGSNOR also has an important function
in plant development and abiotic stress.
While the genetics of tomato (Solanum lycopersicum) GSNOR
(SlGSNOR) have largely been unexplored, the crystal structure
for the corresponding enzyme has been solved to 1.9Å reso-
lution (Kubienová etal., 2013), being the rst plant GSNOR
to be structurally determined. Here, taking a genetic approach,
we uncover key roles for SlGSNOR in plant development,
fruit formation, and immunity in tomato. Collectively, these
data imply that the function of GSNOR is conserved across
plant species. Further, manipulating levels of GSNOR expres-
sion may provide novel mechanisms for the incorporation of
disease resistance and advantageous developmental traits into
crop plants.
Materials and methods
DNA constructs
Tomato GSNOR (Solyc09g064370, http://solgenomics.net/
locus/34669/view) was amplied using SlGSNOR-PstI-F and
SlGSNOR-NotI-R primers (Supplementar y Table S1 at JXB online) and
cloned behind the CaMV2x35S promoter in the pGreenI0029 binary
expression vector to make the GSNOR overexpression (OE) DNA con-
struct. For the SlGSNOR-RNAi DNA construct, sense and antisense
DNA fragments of 369bp were amplied using SlGSNORS-XhoI-F and
SlGSNORS-KpnI-R for the sense fragment, and SlGSNORA-ClaI-F
and SlGSNORA-XbaI-R for the antisense fragment (Table 1), and cloned
in the pHANNIBAL intermediate vector separated by a pDK intron to
make the CaMV35S:sense:intron:antisense:terminator RNAi cassette.
The cassette was then transferred to the pGreenI0029 binary vector.
RNA extraction was performed using an RNeasy Mini Kit (Qiagen)
according to the manufacturer’s instructions. Atotal of 1 µg of RNA
was reverse transcribed to synthesize cDNA using the Omniscript RT
kit (Qiagen) according to the manufacturer’s instructions. A1µl aliquot
of this cDNA was subsequently used in a semi-quantitative PCR for the
amplication of DNA fragments. All DNA fragments were amplied in
a 40μl reaction using Phusion® High Fidelity DNA polymerase (New
England Biolabs). Both the OE and RNAi SlGSNOR DNA constructs
were conrmed by colony PCR and sequenced before transformation in
tomato cultivar MicroTom.
Tomato transformation
The SlGSNOR-OE and SlGSNOR-RNAi DNA constructs were
transformed into tomato cv. MicroTom. Briey, seeds of tomato variety
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GSNOR function in tomato | 4879
MicroTom were surface sterilized in 70% ethanol for 40s, rinsed with
sterile distilled water, kept in 40% sodium hypochlorite (NaOCl)+3
drops/100 ml Tween-20, for 15 min, and rinsed with sterile distilled
water ve times. The seeds were germinated on germination medium
[1/2 MS (Murashige and Skoog, 1962) salts and vitamins, 3% sucrose,
0.8% agar] in the dark at 25±2°C for 7 d.Single Agrobacterium tumefaciens
colonies carrying SlGSNOR-OE and SlGSNOR-RNAi constructs were
grown at 28°C with shaking at 250 rpm for 24h. The cultures were
centrifuged at 12 000rpm for 10 min and bacteria were washed and
re-suspended in liquid MS medium to OD600=0.7. MicroTom explants
were prepared by cutting both ends of the cotyledons and dipped in
A. tumefaciens SlGSNOR-OE and SlGSNOR-RNAi suspension cul-
tures for 5min. The explants were then incubated on shoot induction
medium [SIM: 1/2 MS salts and vitamins, 3% sucrose, 0.8% agar, 2mg
l–1 6-benzylaminopurine (BA), 0.01mg l–1 indole-3-butyric acid (IBA)]
for 2 d and rinsed with sterile distilled water. The explants were then
screened on SIM plates containing 25 mg l–1 kanamycin+500 mg l–1
cefotaxime (transgenic shoot selection medium: TSM). Transgenic shoots
were transferred to fresh TSM every 4 weeks to ensure stringent selection.
Transgenic shoots (~0.5cm) were then transferred to transgenic shoot
elongation medium (TEM: MS salts and vitamins, 3% sucrose, 0.8% agar,
30mg l–1 kanamycin+500mg l–1 cefotaxime) at 25±2 °C in the light.
Plants were transferred to fresh TEM every 3–4 weeks and then to sterile
soil in pots after rooting. Homozygous transgenic plants were obtained as
described previously (Harrison etal., 2006).
Growth of tomatoplants
Seeds from wild-type (WT) and transgenic plants were germinated ei-
ther on 1/2 MS medium (1.1g of MS salt and 5g of sucrose dissolved
in 300ml of water at pH 5.7–5.9 and volume adjusted to 500ml after
adding 4g of agar) or a special peat-based UC (University of California)
compost [100 litres of medium grade peat (Sinclair Horticulture), 25
litres of horticultural sand, 375g of garden limestone (J. Arthur Bowers),
150g of Osmocote Exact 3–4 months (Scotts) and 3 g of Intercept-
70WG (Scotts)]. Seedlings were transplanted to new pots 1 week after
germination and grown at 21°C under long days (16h light/8h dark)
at 800 µmol m−2 s−1 PAR (photosynthetically active radiation) light
intensity.
Growth and inoculation of PstDC3000
Pseudomonas syringae pv. tomato DC3000 (PstDC3000) was grown in LB
liquid medium [tryptone 10 g l–1, yeast extract (Oxoid) 5g l–1, NaCl
(VWR, UK) 10g l–1], with 50µg ml–1 rifampicin at 28°C overnight.
Cells were pelleted by centrifugation before re-suspension in 10 mM
MgCl2. Plants were inoculated as described by Mudgett and Staskawicz
(1999) with some modication. Three-week-old plants were sprayed with
a PstDC3000 virulent suspension with cell density adjusted to 2×108 cfu
ml–1 at OD600. Plants were kept covered inside plastic bags under high
humidity for 24h to allow the opening of stomata for successful bacterial
entrance/inoculation.
PstDC3000 colonycounts
PstDC3000 was inoculated to the tomato WT and transgenic plants as de-
scribed above. The plants were examined for disease symptoms at regular
intervals. Leaf samples were collected at 0, 2, and 4days post-infection
(DPI). Using 12 plants per line, three leaf discs (1cm2) were collected per
plant. Each leaf disc was ground in a microfuge tube in 500µl of 10mM
sterile MgCl2 using a tissue lyser (Qiagen/Retsch) for 2min at 30 shakes
per second. A200µl aliquot of the bacterial suspension was transferred to
a new microfuge tube, and serial dilutions were made to 10–2. After the
serial dilution, 10µl of each dilution was plated on NYG plates [Bacto
peptone 5g l–1, yeast extract (Oxoid) 3g l–1, glycerol (Fisher Scientic)
20ml l–1, Bacto agar 15g l–1] containing 50µg ml–1 rifampicin. The plates
were incubated for 2 d at 28°C and the number of bacterial colonies for
each sample counted and recorded in the best countable dilution. The ex-
periment was repeated three times.
PR1 gene expression
Leaf samples collected for colony counts, from PstDC3000-infected WT
and transgenic plants at 0, 2, and 4 DPI, were used to cut 1cm2 leaf discs
for colony count assay. The rest of the leaf samples were used to extract
RNA for PR gene expression analysis. RNA was isolated from plant sam-
ples as described earlier, and quantication of reverse transcription–PCR
(RT–PCR) was carried out to check PR1 expression in response to in-
fection. Tomato actin was used as a reference gene. Primers for tomato
PR1 and actin genes are given in Supplementary Table S1.
Salicylic acid measurement
Free and conjugated endogenous SA levels were determined using HPLC, as
described by Aboul-Soud etal. (2004) with minor modications. A200mg
aliquot of leaf tissue per sample was collected and promptly frozen in liquid
nitrogen. Samples were then ground in liquid nitrogen using a mortar and
pestle, and transferred to a 2ml microfuge tube, followed by the addition of
1ml of 90% methanol (Fisher Scientic), and vortexed for 1min, before the
sample thawed. The sample was then centrifuged at 15000 g for 5min and
the supernatant transferred to a new tube. The pellet was re-extracted in 1ml
of 100% methanol, centrifuged, and the two supernatants pooled together
and dried in a speed vacuum centrifuge (Speed Vac DNA110, Savant) at me-
dium temperature. The residue resulting from drying the supernatant was then
re-suspended in 1ml of 5% trichloroacetic acid, followed by the addition
of 1ml of ethyl acetate:cyclopentane (Fluka):isopropanol (Fisher Scientic)
(50:50:1) and vortexed for 1min. The organic phase was transferred to a new
tube. The aqueous phase was re-extracted with another 1ml of the organic
50:50:1 mix, and the two supernatants pooled together and evaporated under
heat in the vacuum centrifuge. The aqueous phase was then acidied to pH 1
by addition of 50µl of absolute HCl, boiled for half an hour to release SA from
any acid-labile conjugated forms, and extracted with the organic mix twice.
The two supernatants were pooled together and dried in the vacuum centri-
fuge. The residues were dissolved in 100μl of 100% methanol before 100μl of
H2O was added to give a nal 50% (v/v) methanol concentration. The sam-
ples were ltered through a 0.25μM lter (Millex-GP, Millipore Corporation,
Billerica, MA, USA) and subjected to HPLC analysis. Samples were taken at 4
DPI. SA samples of 1mM and 10mM were used as standard.
Results
SlGSNOR depletion negatively affects seed
development and germination but not root
development
After the transformation of SlGSNOR-OE and SlGSNOR-
RNAi constructs in tomato (cv. MicroTom), SlGSNOR-RNAi
and SlGSNOR-OE lines were generated. RT–PCR results
showed a signicant reduction in SlGSNOR expression in the
RNAi lines, whereas a signicant increase in SlGSNOR expres-
sion was observed in the OE lines (Fig. 1A). Asignicant impact
of SlGSNOR knockdown on the germination percentage was
observed in the RNAi lines, with >80% reduction in germin-
ation (Fig. 1B). This shows that the accumulation of SlGSNOR1
transcripts is tightly regulated in tomato MicroTom and a signi-
cant reduction in its expression leads to lethality, as SlGSNOR-
RNAi lines with greater than ~60% reduction in SlGSNOR1
expression were not viable (Fig. 1C). Both SlGSNOR-RNAi
and SlGSNOR-OE lines conveyed signicant eects on the
overall development of tomato plants, ranging from seed ger-
mination to fruiting and net yield per plant. Reduced GSNOR
expression in SlGSNOR-RNAi lines drastically aected seed
development and reduced the number of seeds produced in
the fruits of the resulting transgenic plants. In representative
RNAi lines that could not be maintained, the seeds were small,
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4880 | Hussain etal.
misshapen, and lacked endosperm. Consequently, these seeds
failed to germinate. In a representative fertile SlGSNOR-RNAi
line (2-2-2), seed germination was reduced by 80% relative to
the WT. The SlGSNOR-OE plants, however, also showed an
average reduction of 20% in germination frequency (Fig. 1B),
although OE plants produced normal healthy seeds (Fig. 1C).
Counterintuitively, the SlGSNOR-RNAi plants also showed
faster germination as compared with WT and SlGSNOR-OE
plants on either MS medium or soil, and showed the appearance
of fresh green tissues at least 1 d before the WT and OE plants.
Suppression of SlGSNOR in tomato did not seem to have
a major impact on the root system of plants under optimal en-
vironmental conditions. The root length of 1- and 5-week-old
plants was analysed (Fig. 2A, B). Root length in SlGSNOR-
RNAi plants was not signicantly dierent from that of WT
plants. In a similar fashion, root length in SlGSNOR-OE plants
was also not visibly dierent from that of WT plants (Fig. 2A,
B). Quantitative analysis and associated statistical testing con-
rmed that the root length of SlGSNOR-RNAi plants and
SlGSNOR-OE plants is not statistically dierent from that of
the WT (Fig. 2C). Collectively, our data imply that reducing
SlGSNOR gene expression impacts tomato seed develop-
ment and germination, but not root development. In contrast,
increasing SlGSNOR expression does not impact either seed
development, seed germination, or root development.
SlGSNOR is required for leaf development
Multiple developmental phenotypes were found to be dif-
ferent in the transgenic plants as compared with WT plants.
Therefore, the average leaf area for WT, SlGSNOR-RNAi, and
SlGSNOR-OE tomato plants was calculated using 5-week-old
plants. Leaf area was measured as an average of multiple leaves of
dierent sizes, ranging from the smallest to the largest and from
the bottom to the top of the plants. The smallest average leaf area
of 468.49mm2 was recorded for SlGSNOR-RNAi plants, com-
pared with 734.35mm2 for the WT. Tomato transgenic plants
overexpressing SlGSNOR had the largest leaves, with an area of
783.10mm2 (Fig. 3A, B) as compared with leaves of WT plants,
although the dierence was not statistically signicant.
SlGSNOR regulates fruit production and flower
development
WT and transgenic SlGSNOR-RNAi and SlGSNOR-OE
tomato plants were analysed for their respective yields. Both
the SlGSNOR-RNAi and SlGSNOR-OE plants showed re-
duced yield per plant as compared with the WT. However,
SlGSNOR-RNAi plants produced an average of 73.56 g of
fruit per plant as compared with 63.96g for SlGSNOR-OE and
179.10g for WT plants (Fig. 4A, B). However, fruits produced
by the SlGSNOR-OE plants were signicantly larger in size
as compared with those of WT plants, whereas those produced
by the SlGSNOR-RNAi plants were smaller in size (Fig. 4B) .
Consistent with the atgsnor1 Arabidopsis mutant, the tomato
SlGSNOR-RNAi plants produced misshapen owers with car-
pels beyond the reach of stamens, which presumably negatively
impacted self-fertilization (Fig. 4C–H). In contrast, SlGSNOR
overexpression had no eect on the oral phenotype; these lines
resembled WT plants with respect to these traits (Fig. 4C–H).
Fig. 1. SlGSNOR suppression negatively affects seed development and germination. (A) Quantification of RT–PCR results showing SlGSNOR transcript
levels in representative tomato WT, RNAi, and OE plants. (B) Percentage germination of tomato WT, RNAi, and OE seeds. Ahighly significant reduction
in germination frequency was recorded for the seeds produced by RNAi plants as compared with WT plants. (C) Phenotype of the WT, RNAi, and
OE seeds. SlGSNOR-RNAi plants produced misshapen, small, and deformed seeds as compared with WT and OE plants. Reduction of SlGSNOR
expression by up to ~60% resulted in lethality and the seeds could not germinate, However, the OE plants produced seeds with a normal phenotype.
Statistical analyses were performed through one-way ANOVA test at a 95% level of confidence. Statistically significant differences are shown by an
asterisk (*). Error bars represent the SD. (This figure is available in colour at JXB online.)
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GSNOR function in tomato | 4881
Overexpression of SlGSNOR promotes resistance to
bacterial pathogens
The tomato cv. MicroTom is not well characterized with respect
to microbial pathogens. The well-characterized leaf pathogen,
PstDC3000, is thought to be an opportunistic pathogen of
this tomato cultivar, with little increase in growth over time
(Takahashi et al., 2005). Three-week-old WT, SlGSNOR-
RNAi, and SlGSNOR-OE plants were spray inoculated with
a PstDC3000 suspension of 2×108 cfu ml–1 in 10mM MgCl2
and 0.02% Silwet L77. This ensured even application of in-
oculum and avoided potential injury. Development of disease
symptoms was monitored daily. Disease symptoms appeared in
SlGSNOR-RNAi plants after 7 d.Chlorosis, the appearance
of typical dark brown lesions surrounded by chlorotic areas,
was clearly visible, especially near leaf margins, on the leaves
of SlGSNOR-RNAi plants. These symptoms are typical of
PstDC3000 infection in susceptible tomato plants. In contrast,
WT and SlGSNOR-OE plants showed delayed and reduced
symptom development (Fig. 5A). Leaf samples were collected
from the inoculated plants at 2 and 4 DPI for the determin-
ation of bacterial titre. SlGSNOR-RNAi plants supported
an increased bacterial titre relative to WT plants at both time
Fig. 2. SlGSNOR suppression does not negatively impact root development. No significant differences were found between the root length of WT, RNAi,
and OE plants after 1 week (A) or 5 weeks of growth (B). Root length measurements of WT, RNAi, and OE plants were statistically analysed using a two-
way ANOVA test at a 95% level of significance. Error bars represent the SD. (This figure is available in colour at JXB online.)
Fig. 3. SlGSNOR suppression reduces average leaf area in tomato. (A) Scanned image showing the arrangement of WT, SlGSNOR-RNAi, and
SlGSNOR-OE plant leaves along with a UK 10 pence coin as a scale marker. (B) Significantly reduced average leaf area was recorded for the RNAi plants
compared with WT and OE plants. However, the increase in the leaf area for OE plants compared with the WT was not significant. Statistical analysis was
performed using one-way ANOVA with 95% confidence Statistical analyses were performed through one-way ANOVA test at a 95% level of confidence.
Statistically significant differences are shown by an asterisk (*). Error bars represent the SD.
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4882 | Hussain etal.
points. Conversely, the number of bacteria in SlGSNOR-OE
plants was signicantly reduced relative to the WT (Fig. 5B).
Thus, reduction of SlGSNOR expression promotes enhanced
disease susceptibility. In contrast, increased SlGSNOR ex-
pression enhances disease resistance against PstDC3000. Thus,
modulation of SlGSNOR transcript abundance impacts the
level of basal disease resistance.
In Arabidopsis, GSNOR has been shown to be a positive
regulator of both SA synthesis and associated signalling (Feechan
etal., 2005; Tada etal., 2008). Therefore, to explore the molecular
mechanism underpinning the regulation of basal disease resist-
ance by SlGSNOR, we rst determined the levels of SA in WT,
SlGSNOR-RNAi, and SlGSNOR-OE plants. The basal and
pathogen-induced levels of SA in SlGSNOR-RNAi plants were
55% and 57.74%, respectively, of those present in WT plants.
The concentrations of SA found in the GSNOR-overexpressing
plants were 360% and 132.8% higher than those in the WT be-
fore and after infection, respectively (Fig. 5C).
Presumably, these changes in the levels of SA impact the ex-
pression of SA-dependent genes, including the well-established
SA marker gene, PR1 (Uknes etal., 1992). We therefore deter-
mined the level of PR1 gene expression in WT, SlGSNOR-
RNAi, and SlGSNOR-OE plants in response to pathogen
challenge. Reduced PR1 transcript accumulation was observed
in SlGSNOR-RNAi lines 2 and 4 DPI with respect to the
WT. Conversely, PR1 gene expression was signicantly in-
creased in SlGSNOR-OE plants at 2 and 4 DPI relative to the
WT (Fig. 5D).
Discussion
Our data show that depletion of SlGSNOR levels impacts
growth and development of tomato cv. MicroTom. Reduction
of SlGSNOR function results in the loss of apical domin-
ance, changes in leaf shape, perturbations in seed development
and germination, and, signicantly, a reduction in the yield of
Fig. 4. Manipulation of SlGSNOR levels impacts fruit production and flower development. (A) Average yield (g per plant) of WT, SlGSNOR-RNAi, and
SlGSNOR-OE tomato plants. Both SlGSNOR-RNAi and SlGSNOR-OE transgenic lines showed a highly significant reduction in yield. (B) OE plants
produced large, healthy fruits with a good number of healthy viable seeds, while RNAi plants produced small fruits typically <2.5cm in diameter with few
and mostly non-viable seeds. (C–H) SlGSNOR-RNAi plants produced misshapen flowers with long carpels extending beyond the reach of the stamens
(D, G) compared with the WT (C, F) and OE plants (E, H). Statistical analyses were performed through one-way ANOVA test at a 95% confidence level.
Statistically significant differences are shown by an asterisk (*). Error bars represent the SD. (This figure is available in colour at JXB online.)
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GSNOR function in tomato | 4883
tomato fruit. In contrast, overexpression of SlGSNOR has no
impact on the growth of tomato cv. MicroTom. Our ndings
also highlight a key role for SlGSNOR in disease resistance.
Thus, depletion of SlGSNOR levels resulted in enhanced
disease susceptibility to the bacterial pathogen, PstDC3000.
Conversely, overexpression of SlGSNOR promoted disease re-
sistance due to increased SA accumulation and associated ex-
pression of SA-dependentgenes.
While AtGSNOR has been extensively studied in
Arabidopsis, there been no previous information on the
genetics of GSNOR in crop plants. In contrast, the eect
of NO on seed germination, root architecture, and fruit
ripening has been studied employing NO donors in crop
plants (Zandonadi etal., 2010; Semchuk etal., 2011). Analysis
of GSNO in the main organs of pepper plants established
that this metabolite was most abundant in roots, followed by
leaves and stems. These ndings directly correlated with the
content of NO in each organ and inversely correlated with
GSNOR activity (Airaki etal., 2011). Subcellular localization
of GSNO in pea leaves established the presence of GSNO
in the cytosol, chloroplasts, mitochondria, and peroxisomes
(Barroso etal., 2013). While these studies have provided excel-
lent and compelling circumstantial evidence for a key role for
GSNO and, by extension, GSNOR in plant developmental
processes in crop plants, direct genetic evidence has not been
established. Our data show that depletion of SlGSNOR does
indeed impact a number of development processes outside
the model plant, Arabidopsis. Thus, reduction of SlGSNOR
transcript accumulation decreases individual leaf size and con-
sequently total leaf area. Seed size is also reduced and seed
Fig. 5. Overexpression of SlGSNOR promotes disease resistance. (A) Development of disease symptoms in the stated lines after 1 week of infection
with PstDC3000. Yellow chlorotic areas surrounding dark brown lesions can be seen on the leaves of RNAi plants. (B) Graph showing bacterial growth
after 2 d and 4 d of infection. Significantly higher bacterial growth was observed in RNAi plants, whereas the OE plants supported significantly lower
bacterial growth after 2 d and 4 d of infection. (C) Total salicylic acid (SA) levels were measured in unchallenged leaves, in addition to plants infected with
PstDC3000 after 4 d of infection. SlGSNOR-OE plants showed a significantly higher level of basal and induced SA compared with WT plants. On the
other hand, the RNAi plants produced lower quantities of SA both before and after infection. (D) Expression of the SA marker gene, PR1, was found to
be significantly higher in the OE plants after 2 d and 4 d of infection by PstDC3000 as compared with that in WT plants. RNAi plants showed significantly
lower PR1 expression compared with WT plants. Statistical analyses were performed using two-way ANOVA test at a 95% level of confidence.
Statistically significant differences are shown by an asterisk (*). Error bars represent the SD. (This figure is available in colour at JXB online.)
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4884 | Hussain etal.
from SlGSNOR-RNAi plants exhibit a lower germination
frequency. Signicantly, fruit size and total fruit yield are also
reduced.
NO bioactivity has been strongly linked to plant repro-
ductive biology (Bright etal., 2009; Zafra etal., 2010). Thus, NO
can act as a negative regulator of pollen tube growth in plants
such as Lilium longiorum, Arabidopsis, and Paulownia tomentosa
(Prado etal., 2004, 2008; He etal., 2007). Conversely, NO has
been reported as a positive stimulus of pollen tube growth in
Pinus bangeana, functioning in a dose-dependent manner (Y.
Wang etal., 2009). In SlGSNOR-RNAi plants, our data show
that the structure of the reproductive organs was impacted;
these lines developed long carpels, resulting in the stigma being
spatially removed from the surrounding anthers, decreasing
pollen transfer and, by extension, self-fertility. These pheno-
types parallel those observed in Arabidopsis plants possessing
null mutations in AtGSNOR (Kwon etal., 2012), implicating
conservation of GSNOR function from Arabidopsis to to-
mato across a number of developmental processes with vis-
ible outcomes. Interestingly, null mutations in Arabidopsis
AtGSNOR also perturb root development, resulting in shorter
roots. However, in contrast, depletion of SlGSNOR transcripts
did not visibly impact root development. Perhaps sucient
SlGSNOR activity was still present in the relevant root cells
of these plants to enable the completion of key growth and/or
developmental processes in this organ. Alternatively, a role for
GSNOR function in root development may not be conserved
between Arabidopsis and tomato. GSNOR is a single-copy
gene in both tomato and Arabidopsis. Our ndings suggest that
strong reduction of SlGSNOR expression resulted in the for-
mation of non-viable seeds. Thus, null mutants of SlGSNOR
might not be maintained in tomato cv. MicroTom and perhaps
other tomato cultivars.
Ripening of both climacteric (e.g. tomato) and non-
climacteric (e.g. pepper) fruits is another area where NO func-
tion and associated S-nitrosylation have been explored (Corpas
etal., 2018). Climacteric fruits continue ripening after being
picked, a process accelerated by ethylene. Non-climacteric
fruits can ripen only when still attached to their respective
plant. These fruits have a short shelf-life if harvested when ripe.
The application of NO gas or NO donors to a number of dif-
ferent climatic fruits has been shown to delay fruit ripening.
In this context, NO can repress both ethylene metabolism
and signalling, while simultaneously inducing antioxidative
enzymes, which are thought to prevent oxidative damage.
Intriguingly, NO gas has also been shown to delay fruit
ripening in non-climacteric fruits and, in addition, increase the
amount of ascorbate (vitamin C) (Rodríguez-Ruiz etal., 2017;
Corpas etal., 2018). Thus, NO treatment of non-climatic fruits
may convey dual advantages: extending both fruit shelf-life and
quality. This exciting research has clearly uncovered a potential
biotechnological application of NO (Manjunatha etal., 2010;
Corpas etal., 2018). Our ndings suggest that continuous de-
pletion of SlGSNOR function and, by extension, increasing
GSNO and associated global S-nitrosylation, both decreases
the size of individual fruits and reduces the overall fruit yield.
It will now be interesting for future studies to explore the bio-
chemical composition and shelf-life of these fruits. Therefore,
to maximize the potential utility of NO to augment both fruit
shelf-life and quality, insights into the molecular mechanisms
whereby NO and cognate S-nitrosylation control these pro-
cesses will be important, because our data suggest that too
much NO/GSNO can have adverse eects on both individual
fruit size and totalyield.
The role of GSNO and NO during immunity in crop plants
remains relatively unclear, because a genetic analysis has not
complemented the biochemical studies to date. Two sunower
(Helianthus annuus L.) cultivars either resistant or susceptible to
infection by the downy mildew pathogen, Plasmopara halstedii,
were employed to investigate the role of GSNO and related
reactive nitrogen intermediates (RNIs) in the immune re-
sponse of this plant. In the susceptible cultivar, an increase in
both protein tyrosine nitration and SNOs was detected, inde-
pendent of NO generation, suggesting that microbial patho-
gens induce nitrosative stress in susceptible sunower cultivars.
Conversely, in the resistant cultivar, there was no increase in
either protein tyrosine nitration or SNOs, implying an absence
of nitrosative stress. Therefore, protein tyrosine nitration might
mark nitrosative stress in plants during microbial infection
(Chaki etal., 2009). In potato, after challenge with an avirulent
Phytophthora infestans isolate, relatively high levels of GSNO and
SNOs concentrated in the main vein of potato leaves, implying
a possible mobile function of these compounds in the transfer
of NO bioactivity. In contrast, during a virulent P. infestans in-
fection, low-level production of NO and ROIs occurred; it was
proposed that this might result in the delayed up-regulation of
PR genes and the subsequent compromised resistance towards
this pathogen (Arasimowicz‐Jelonek etal., 2016). Moreover, in
lettuce (Lactuca sativa), a GSNOR-mediated decrease of SNOs
was found to be a general feature of lettuce responses to both
downy and powdery mildew infection, while resistance to
Bremia lactucae, the causal agent of lettuce downy mildew, was
found to parallel an increase of GSNOR activity. Thus, modu-
lation of GSNOR activity appears to play a key role in lettuce–
mildew interactions (Tichá etal., 2018).
In Arabidopsis, loss-of-function mutations in AtGSNOR
result in an increase in total S-nitrosylation and a reduction
in SA biosynthesis and associated signalling, compromising
disease resistance (Feechan etal., 2005; Tada etal., 2008; Yun
et al., 2016). In complete contrast, depletion of AtGSNOR
transcripts has been reported to result in disease resistance
(Rustérucci etal., 2007). This may reect the complex role of
(S)NO in plant immunity. Thus, depleting AtGSNOR levels
may increase SNO levels less relative to a null mutation in
AtGSNOR and this dierence in relative SNO concentrations
may result in dierent immune ouputs (i.e. resistance versus
susceptibility, respectively). If this posit is correct, our deple-
tion of SlGSNOR transcripts to a similar extent in tomato
might be predicted to lead to increased disease resistance. Our
ndings show that depletion of GSNOR function in tomato
results in decreased SA biosynthesis and signalling, leading to
compromised basal resistance. This surprisingly contrasts with
previous data showing that depletion of AtGSNOR transcripts
results in disease resistance (Rustérucci etal., 2007); however,
it is similar to other data proposing that null mutations in
AtGSNOR compromise plant immunity (Feechan etal., 2005;
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GSNOR function in tomato | 4885
Tada etal., 2008; Yun et al., 2011). Therefore, it appears un-
likely that these previous ndings (Rustérucci et al., 2007)
can be explained by dierences in relative SNO concentra-
tions between AtGSNOR-depleted lines and those possessing
null mutations in AtGSNOR (Feechan etal., 2005; Tada etal.,
2008; Yun etal., 2011).
Importantly, our ndings show that the function of GSNOR
in disease resistance appears to be conserved from Arabidopsis
to tomato. Signicantly, the overexpression of AtGSNOR
in Arabidopsis conveyed broad-spectrum disease resistance,
without constitutive SA accumulation or associated signalling
(Feechan etal., 2005). Rather, AtGSNOR overexpression sup-
ported a potentiation of SA-dependent gene expression fol-
lowing attempted pathogen infection. Moreover, this resistance
was not associated with a negative impact on growth or a yield
penalty under laboratory conditions (Feechan etal., 2005), sug-
gesting that manipulation of GSNOR activity might provide
a novel mechanism to convey broad-spectrum disease resist-
ance in crop plants. Our data suggest that the overexpression
of SlGSNOR also does not negatively impact growth, but it
does decrease total fruit yield. However, on the positive side,
overexpression of SlGSNOR signicantly increased the size of
individual tomato fruits, which might be attractive for some
markets. Therefore, manipulating SlGSNOR expression via
traditional crop breeding or gene editing approaches might
provide novel strategies to convey disease resistance and per-
haps also modulate the properties of tomato fruits.
Supplementarydata
Supplementary data are available at JXB online.
Table S1. List of primers used in RT–PCR.
Acknowledgements
AH was supported by Higher Education Commission (HEC) Pakistan
for PhD Studentship. Research in the laboratory of GJL has been sup-
ported by BBSRC grant BB/DO11809/1.
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