Nitric Oxide, Ethylene, and Auxin Cross Talk Mediates

Greening and Plastid Development in Deetiolating

Tomato Seedlings1[OPEN]

Nielda K.G. Melo, Ricardo E. Bianchetti, Bruno S. Lira, Paulo M.R. Oliveira, Rafael Zuccarelli,
Devisson L.O. Dias, Diego Demarco, Lazaro E.P. Peres, Magdalena Rossi, and Luciano Freschi*
Department of Botany, University of São Paulo, Sao Paulo 05508–090, Brazil (N.K.G.M., R.E.B., B.S.L., P.M.R.O.,
R.Z., D.L.O.D., D.D., M.R., L.F.); and Department of Biological Sciences, Escola Superior de Agricultura Luiz de
Queiroz, University of São Paulo, Piracicaba 13418–900, Brazil (L.E.P.P.)
ORCID IDs: 0000-0002-0820-6682 (B.S.L.); 0000-0003-0912-9065 (R.Z.); 0000-0002-3230-6544 (D.L.O.D.); 0000-0002-8761-5934 (L.E.P.P.);
0000-0003-3650-772X (M.R.); 0000-0002-0737-3438 (L.F.).

The transition from etiolated to green seedlings involves the conversion of etioplasts into mature chloroplasts via a multifaceted,
light-driven process comprising multiple, tightly coordinated signaling networks. Here, we demonstrate that light-induced
greening and chloroplast differentiation in tomato (Solanum lycopersicum) seedlings are mediated by an intricate cross talk
among phytochromes, nitric oxide (NO), ethylene, and auxins. Genetic and pharmacological evidence indicated that either
endogenously produced or exogenously applied NO promotes seedling greening by repressing ethylene biosynthesis and
inducing auxin accumulation in tomato cotyledons. Analysis performed in hormonal tomato mutants also demonstrated that
NO production itself is negatively and positively regulated by ethylene and auxins, respectively. Representing a major
biosynthetic source of NO in tomato cotyledons, nitrate reductase was shown to be under strict control of both phytochrome
and hormonal signals. A close NO-phytochrome interaction was revealed by the almost complete recovery of the etiolated
phenotype of red light-grown seedlings of the tomato phytochrome-deficient aurea mutant upon NO fumigation. In this mutant,
NO supplementation induced cotyledon greening, chloroplast differentiation, and hormonal and gene expression alterations
similar to those detected in light-exposed wild-type seedlings. NO negatively impacted the transcript accumulation of genes
encoding phytochromes, photomorphogenesis-repressor factors, and plastid division proteins, revealing that this free radical can
mimic transcriptional changes typically triggered by phytochrome-dependent light perception. Therefore, our data indicate that
negative and positive regulatory feedback loops orchestrate ethylene-NO and auxin-NO interactions, respectively, during the
conversion of colorless etiolated seedlings into green, photosynthetically competent young plants.

Chloroplast biogenesis and maturation are critical for plastid progenitor known as proplastid or from the
plant growth and development, because this organelle dark-grown transitory form, the etioplast (Pogson and
is responsible for photosynthesis and many other es- Albrecht, 2011). Considering the fact that under most
sential metabolic pathways. During seedling develop- natural conditions light is unavailable or insufficient
ment, chloroplasts may differentiate directly from the during the initial development of germinating seed-
lings, the formation of etioplasts and their subsequent
1
differentiation into chloroplasts is of high adaptive
This work was supported by the Conselho Nacional de Desen-
value for most, if not all, terrestrial seed plants. Fa-
volvimento Científico e Tecnológico (grant nos. 472669/2010–9 and
442045/2014–0 to L.F.) and the São Paulo Research Foundation (grant
cilitating the prompt differentiation into functional
no. 2013/18056–2 to L.F.). chloroplasts, etioplasts typically accumulate thyla-
* Address correspondence to [email protected]. koid lipids and large amounts of the chlorophyll pre-
The author responsible for distribution of materials integral to the cursor protochlorophyllide (Pchlide) bound to Pchlide
findings presented in this article in accordance with the policy de- NADPH-oxidoreductase, forming a semicrystalline,
scribed in the Instructions for Authors (www.plantphysiol.org) is: membranous structure known as prolamellar body (PLB;
Luciano Freschi ([email protected]). Von Wettstein et al., 1995).
N.K.G.M. conducted most of the experiments; R.E.B., B.S.L., and Unarguably, light perception and signaling repre-
M.R. participated in the gene expression experiments; P.M.R.O. per- sent the master switch for the etioplast-to-chloroplast
formed the hormonal analysis; D.D. participated in the ultrastructure
conversion, controlling the transcription of regulatory
analysis; R.Z. and D.L.O.D. conducted part of the experiments; L.E.P.P.
produced the transgenic lines and mutants and participated in the
genes, whose products regulate the synthesis of chlo-
interpretation of results; L.F. conceived the project and supervised the rophylls and photosynthetic apparatus components,
experiments; N.K.G.M. and L.F. wrote the article with contributions ultimately leading to the final structural configuration
from the other authors. and biochemical composition of mature chloroplasts
[OPEN]
Articles can be viewed without a subscription. (Waters and Langdale, 2009). Among these light-
www.plantphysiol.org/cgi/doi/10.1104/pp.16.00023 induced elements, GOLDEN2-LIKE (GLK ) genes,
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 NO-Hormone Cross Talk during Light-Evoked Greening

which encode GARP transcription factors, are known to HP1/DDB1, HP2/DET1, COP1, and HY5, has emerged
coordinate the expression of many photosynthesis- as an efficient strategy to improve plastidial biogenesis
related genes, promoting chloroplast maturation and and development and, consequently, the nutritional
maintenance (Waters et al., 2009; Powell et al., 2012). quality of tomato fruits (Liu et al., 2004; Davuluri et al.,
The shift from skotomorphogenic to photomorpho- 2005; Wang et al., 2008).
genic development relies on the combined action of As explained above, the role of photoreceptors and
phytochromes and cryptochromes, which are the two light signal transduction proteins in chloroplast devel-
photoreceptor families mainly responsible for gene ex- opment has been explored extensively in different plant
pression reprogramming in deetiolating seedlings (Jiao models. In contrast, few studies have focused on the
et al., 2007; López-Juez et al., 2008; Franklin and Quail, interplay between photoreceptors and other endoge-
2010). Photoactive holophytochromes are formed when nous signals, such as plant hormones, during the
phytochrome apoproteins, encoded by a small nuclear coordination of plastid differentiation with plant
gene family, become covalently linked to the invariant development and environmental stimuli (Egea et al.,
linear tetrapyrrole chromophore phytochromobilin. 2010). In this context, an antagonistic relationship be-
Therefore, due to the loss of function of all phytochrome tween cytokinins and abscisic acid (ABA) during chlo-
types, the phytochromobilin-deficient mutants usually roplast differentiation in deetiolating seedlings has
exhibit more severe chloroplast differentiation impair- been described (Kusnetsov et al., 1998; Kravtsov et al.,
ment than type-specific phytochrome mutants (Kendrick 2011). Whereas cytokinins activate cotyledon and leaf
et al., 1997). greening by stimulating plastid biogenesis and devel-
Active phytochromes are translocated from cytosol opment, ABA has been shown to have the opposite
to the nucleus, where they bind to phytochrome- effect (Chory et al., 1994; Kusnetsov et al., 1998; Galpaz
interacting factors and target these basic helix-loop- et al., 2008; Kravtsov et al., 2011; Cortleven and
helix transcription factors for degradation (Franklin Schmülling, 2015). In contrast to cytokinins and ABA,
and Quail, 2010; Leivar and Quail, 2011). In Arabi- the influence of auxins and ethylene on plastid bio-
dopsis (Arabidopsis thaliana), losses of particular genesis and etioplast-to-chloroplast differentiation has
phytochrome-interacting factors have been shown to received considerably less attention. However, an inti-
disturb photosynthetic protein accumulation and mate connection between these hormones has been
chloroplast development in both dark- and light-grown reported during other important events related to
seedlings (Leivar and Quail, 2011). Moreover, negative seedling deetiolation, such as the regulation of hypo-
regulators of light signal transduction, such as cotyl elongation and the formation, maintenance, and
CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) or opening of the apical hook in eudicotyledons (Symons
DEETIOLATED1 (DET1), have been identified as sup- and Reid, 2003; Van de Poel et al., 2015). Moreover, the
pressors of chloroplast development both in cotyledons biosynthesis and signaling of these hormones also have
of dark-grown seedlings and in nonphotosynthetic tis- been shown to change significantly upon illumination
sues of light-grown plants (Chory et al., 1989; Chory and (Symons and Reid, 2003; López-Juez et al., 2008; Van de
Peto, 1990; Deng and Quail, 1992). The interaction of COP Poel et al., 2015).
and DET proteins among themselves and with other Besides plant hormones, other endogenous signal
light-regulated proteins, such as UV-DAMAGED DNA- molecules also seem to participate in controlling seed-
BINDING PROTEIN1 (DDB1) and CULLIN4 (CUL4), ling greening. The gaseous free radical nitric oxide
gives rise to the so-called COP9 signalosome, which tar- (NO), for instance, has emerged as a stimulatory signal
gets photomorphogenesis-promoting factors, such as for the acquisition of photomorphogenic traits in dif-
LONG HYPOCOTYL5 (HY5), for proteasomal degra- ferent plant species, regulating processes such as seed
dation (Wei et al., 2008). Therefore, the light-evoked germination, hypocotyl elongation, and cotyledon and
degradation of COP9 signalosome components pro- leaf greening (Beligni and Lamattina, 2000; Zhang et al.,
motes HY5 accumulation and, consequently, activates 2006; Lozano-Juste and León, 2011). During seedling
the expression of photomorphogenesis-related genes greening, NO seems to intensify the responses to light
(Wei et al., 2008). stimuli, promoting the accumulation of chlorophylls and
As in Arabidopsis, orthologs of DET1, COP1, DDB1, chloroplast-related proteins (Beligni and Lamattina,
and HY5, as well as other light signaling components, 2000; Zhang et al., 2006). Moreover, increased NO
also have been demonstrated to control plastid biogene- production also has been reported in deetiolating
sis and development in leaf and fruit tissues of tomato Arabidopsis and wheat (Triticum aestivum) seedlings
(Solanum lycopersicum; Liu et al., 2004; Davuluri et al., (Lozano-Juste and León, 2011; Liu et al., 2013).
2005; Kolotilin et al., 2007; Wang et al., 2008). For in- Although NO biosynthesis in plants is still not fully
stance, mutations in HIGH PIGMENT1 (HP1) and HP2, characterized, nitrate reductase (NR) has been consid-
which are the orthologs of AtDDB1 and AtDET1, re- ered one of the most likely candidates responsible for
spectively, render plants with increased chlorophyll and NO production under physiologically relevant condi-
carotenoid content as well as higher chloroplast number tions (Planchet and Kaiser, 2006a; Gupta et al., 2011).
and size in both leaf and fruit cells (Mustilli et al., 1999; NR is regulated at both the transcription and post-
Cookson et al., 2003; Kolotilin et al., 2007). More recently, translational levels by multiple environmental and en-
manipulation of light signaling components, such as dogenous factors (Kaiser and Huber, 2001). Light, for
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Melo et al.

instance, is a critical environmental cue that regulates tomato hp2 mutants (Mustilli et al., 1999). The plastid
both NR gene transcription and protein phosphoryla- internal membranous structure observed in the wild
tion (Lillo and Appenroth, 2001). In the presence of type and hp1 under red light (RL) or blue light (BL), or
nitrate, NR transcript and protein levels increase dra- in au seedlings under BL, was virtually indistinguish-
matically in deetiolating seedlings soon after exposure able, with the formation of granal thylakoids, the dis-
to illumination. Apparently, such a response mostly appearance of PLBs, and, in some cases, the presence of
depends on light perception via phytochromes (Becker starch grains. In contrast, chloroplasts of RL-treated au
et al., 1992; Goud and Sharma, 1994), which can be seedlings presented neither granal thylakoids nor PLBs
mimicked or intensified by the exogenous application (Fig. 1). Ultrastructural plastid features observed in
of cytokinins and/or auxins (Lu et al., 1992; Yu et al., cotyledon cells of wild-type, hp1, and au seedlings
1998). As a result, light and plant hormones interact grown under white light resembled those found under
intensively to determine the induction of NR during the BL conditions (Supplemental Fig. S1).
deetiolation process. In cotyledons of wild-type seedlings, maximum
Based on the intertwined NO-hormone signaling chlorophyll and carotenoid levels were observed 48 h
cascades described for numerous plant developmental after the start of either RL or BL treatment, remaining
responses (Freschi, 2013; Sanz et al., 2015), interactions relatively stable thereafter (Fig. 2). The time course of
between NO and plant hormones during seedling pigment accumulation was similar in wild-type and au
greening also might be expected. These NO-hormone seedlings exposed to BL. As expected, RL failed to
cross talks might involve not only the influence of NO induce photosynthetic pigment accumulation in the
on phytohormone metabolism, perception, or signal phytochromobilin-deficient mutant. Consistent with its
transduction but also the modulation of endogenous light-hypersensitive phenotype, hp1 seedlings exhibi-
NO levels by plant hormones (Freschi, 2013). In this ted pigment levels considerably higher than the wild
context, by a dedicated biochemical characterization type under either RL or BL (Fig. 2).
and the use of genetic resources, we investigated the NO levels in cotyledon tissues of dark-grown and
role of NO in plastid development during tomato deetiolating wild-type, hp1, and au seedlings were
seedling deetiolation and the regulatory interplay of determined using the fluorometric quantification
NO with auxins and ethylene. The results showed that method based on the cell-impermeant NO probe
NR-dependent NO production mediates light-evoked diaminorhodamine-4M (DAR-4M; Fig. 2C) and com-
greening by regulating the expression of light signaling pared with the maximum activity (Fig. 2D) and ac-
as well as plastid division and differentiation genes. tivation state (Fig. 2E) of NR. Under continuous
Moreover, our study uncovered a mutual positive and darkness, all genotypes exhibited reduced endogenous
negative feedback between NO-auxin and NO-ethylene NO release associated with extremely low levels of
metabolism, respectively, during light-dependent re- maximum NR activity. In contrast, endogenous NO
sponses. and maximum NR activity dramatically increased in
wild-type seedlings during the first 24 h of RL or BL. In
au seedlings, NO generation increased exclusively un-
RESULTS der BL conditions, correlating with the maximum NR
NR-Derived NO Production Temporally Coincides with activity. The light-hypersensitive mutant hp-1 exhibited
Light-Evoked Greening the highest values of both NO release and maximum
NR activity under either RL or BL, which exceeded by
To gain insight into the possible role of endoge- several times the levels detected in wild-type or au
nous NO in the regulation of etioplast-to-chloroplast seedlings. Regardless of the light treatment, hp-1 seed-
differentiation, the time course of plastid structural lings also exhibited slightly higher values of NR acti-
changes, photosynthetic pigment accumulation, and vation state, suggesting a negative influence of HP1/
NO content fluctuations was evaluated in cotyledons SlDDB1 not only on total NR activity but also on NR
of deetiolating and dark-grown tomato seedlings. posttranslational regulation (Fig. 2).
Wild-type tomato and two contrasting photomor- A concomitant and progressive increase in both
phogenic mutants were compared: (1) the pale-green, chlorophyll and NO levels was observed within the first
phytochromobilin-deficient mutant aurea (au) and (2) 24 h of either RL or BL exposure, with significant in-
the dark-green, light-hypersensitive mutant hp1 (Carvalho creases in both parameters taking place as soon as 6 h of
et al., 2011). light treatment (Fig. 2F; Supplemental Fig. S2). The
Plastids of dark-grown wild-type and au seedlings highest values of NR activity and endogenous NO re-
presented an internal structure typical of etioplasts, lease were observed after 18 and 24 h of light treatment,
exhibiting the typical lattice-like membranous structure respectively. In contrast, maximum chlorophyll and
of PLBs (Fig. 1). In contrast, dark-grown hp1 tomato carotenoid levels were only observed after 48 or 72 h of
seedlings presented semideveloped chloroplasts in- either RL or BL exposure, respectively (Supplemental
stead of etioplasts, exhibiting PLBs converted into Fig. S2). Therefore, whereas the light-driven accumu-
prothylakoid membranes (Fig. 1) and resembling the lation of photosynthetic pigments temporally coincided
plastid structure of dark-grown Arabidopsis cop and det with increases in endogenous NO content within the
mutants (Chory et al., 1989; Deng and Quail, 1992) and first 24 h of light exposure, the maximum accumulation
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 NO-Hormone Cross Talk during Light-Evoked Greening

Figure 1. Plastid structure in cotyledon

cells of tomato photomorphogenic mu-
tants exposed to distinct light conditions.
Wild-type (WT), hp1, and au seedlings
dark grown for 120 h were either kept in
darkness or transferred to continuous red
light (RL) or blue light (BL) treatment for 72 h
before ultrastructural analysis. G, Granal
thylakoid; S, Starch; PLB, prolamellar body.
Bars = 0.5 mm.

of these pigments was clearly preceded by a transitory NO Fumigation Promotes Greening in the
peak in both NR activity and NO production (Fig. 2F; Phytochromobilin-Deficient Tomato Mutant
Supplemental Fig. S2).
This clear temporal correlation among the time To further investigate the causal relationship between
courses of NR activity, NO production, photosynthetic NO and chloroplast differentiation, pharmacology-based
pigment accumulation and plastid development (Figs. experiments involving the continuous fumigation of to-
1 and 2; Supplemental Fig. S2) was taken as a first line of mato seedlings with NO-enriched atmospheres were
evidence suggesting the involvement of NR-dependent performed. Whereas NO fumigation had no marked
NO production in light-evoked greening in tomato impact on chlorophyll and carotenoid levels in dark-
seedlings. To confirm the relevance of NR as a source of or BL-exposed tomato seedlings (Supplemental Fig.
NO in tomato cotyledons, different treatments were S4), a very clear increase in both these pigments was
conducted to inhibit the activity of this enzyme, in- observed when au seedlings were fumigated continu-
cluding the use of a nitrogen-free medium and two ously with 50, 100, or 150 mL L21 under RL (Fig. 3A).
modified Murashige and Skoog (MS) media containing Such NO-induced greening was shown to be dose de-
ammonium or Gln as the sole source of nitrogen. All pendent, since significant increases in chlorophylls
these approaches significantly inhibited both NR ac- and carotenoids were observed as quickly as 24 h after
tivity and reduced the cotyledon NO levels in wild-type simultaneous treatment with RL and 150 mL L21 NO,
seedlings by about 80% under either RL or BL treatment whereas fumigation with 50 or 100 mL L21 NO led to
(Table I). significant increases in these pigments only after 48 h
In plants, the production of NO from Arg via a nitric of treatment. The levels of pigments in au cotyledons
oxide synthase (NOS)-like activity also has been under RL and 150 mL L21 NO (Fig. 3A) were similar to
linked to NO production during some plant devel- those observed in RL-treated wild-type cotyledons
opmental and stress responses (Gupta et al., 2011; (Fig. 2). NO fumigation also induced the partial for-
Mur et al., 2013). However, treatments with the mation of granal thylakoids in RL-treated au seedlings
mammalian NOS inhibitor N G -nitro- L -Arg methyl (Fig. 3B). At the end of the treatment, the chloroplast
ester neither affected NO production nor altered the number per cotyledon cell in au seedlings exposed to
greening process in RL- or BL-exposed wild-type to- RL plus NO fumigation (38.2 6 5.3) was statistically
mato seedlings (Supplemental Fig. S3). Moreover, no similar to that observed in RL-treated wild-type
significant nitrite accumulation was detected in cot- seedlings (36.1 6 4.2). No significant influence of NO
yledons of deetiolating tomato seedlings (data not fumigation on Pchlide content was observed in etio-
shown); therefore, physiologically relevant levels of lated au seedlings (Supplemental Fig. S4); therefore,
nonenzymatic NO production from nitrite seem un- the positive influence of NO fumigation on chloro-
likely (Bethke et al., 2004). phyll accumulation apparently does not involve
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Melo et al.

Figure 2. Light-driven tomato seedling greening temporally coincides with the rise in both NO levels and NR activity. Wild-type (WT), hp1, and au
seedlings dark grown for 120 h were either kept in darkness or transferred to continuous RL or BL treatment. A, Chlorophylls. B, Carotenoids. C,
Fluorometric quantification of endogenous NO release. D, NR maximum activity (NRmax). E, NR activation state. F, Chlorophyll levels, endogenous NO
release, and NR maximum activity in wild-type seedlings within the first 24 h of RL or BL exposure. In F, the maximum chlorophyll, NO, and NR values
were set to 1 for comparison. Values shown are means 6 SE. Asterisks indicate statistically significant differences compared with the wild type (in A–E)
or compared with the dark treatment (in F) at each sampling time (Student’s t test, P , 0.05). DW, Dry weight.

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 NO-Hormone Cross Talk during Light-Evoked Greening

Table I. Maximum and actual NR activity and endogenous NO release in wild-type tomato seedlings cultivated in different sources of nitrogen
under RL or BL
When present in the medium, nitrogen was always available at a final concentration of 30 mM. Values shown are means 6 SE. Statistically sig-
nificant differences between the control and treatments are indicated in boldface (Student’s t test, P , 0.05). n.d., Not detected.
Treatment Maximum NR Activity Actual NR Activity NO Fluorescence
RL BL RL BL RL BL
2 21 21 21 21
mmol NO2 g dry wt h g dry wt h
Control 0.60 6 0.04 0.87 6 0.07 0.27 6 0.03 0.34 6 0.03 76.9 6 15.4 65.5 6 2.9
Nitrogen free 0.05 6 0.01 0.03 6 0.01 0.02 6 0.00 0.01 6 0.00 21.0 6 2.4 16.5 6 0.6
Ammonium 0.05 6 0.01 0.13 6 0.02 0.02 6 0.01 0.04 6 0.01 14.4 6 1.2 14.8 6 1.6
Gln n.d. n.d. n.d. n.d. 11.6 6 1.1 11.4 6 0.9

significant changes in chlorophyll precursor levels prior for SlPHYF, all other genes encoding for phytochrome
to light exposure. apoproteins in tomato (SlPHYA, SlPHYB1, SlPHYB2,
Therefore, NO fumigation partially rescued the se- and SlPHYE; Kendrick et al., 1997) exhibited signifi-
vere defect in greening and chloroplast development cantly higher mRNA levels in au than in wild-type
typically observed in au seedlings grown under mono- seedlings under RL conditions. Interestingly, a pro-
chromatic RL, and such an experimental setup emerged gressive reduction in the transcript levels of SlPHYA,
as a particularly interesting model system to study the SlPHYB1, and SlPHYE, but not in SlPHYB2 and
endogenous mechanisms responsible for converting NO SlPHYF, was observed in RL-treated au seedlings upon
signals into cotyledon greening and chloroplast differ- NO fumigation (Fig. 4A; Supplemental Fig. S5).
entiation. Under continuous RL, mRNA levels of genes en-
coding the photomorphogenic repressor proteins
SlCOP1, SlCUL4, SlDET1, and SlDDB1 were also more
NO Fumigation Alters mRNA Accumulation of abundantly detected in au than in wild-type seedlings,
Photomorphogenesis-Related and Plastid Division and and, quite remarkably, NO fumigation was found to
Differentiation Genes trigger a progressive reduction in the expression of all
these genes. After 72 h of NO treatment, the mRNA
To better understand the mechanisms underlying the level of these genes in au were as low as those observed
NO-dependent rescue of the cotyledon greening defi- in fully deetiolated wild-type seedlings (Fig. 4A;
ciency phenotype in RL-treated au seedlings, a tran- Supplemental Fig. S5).
script profile of genes encoding phytochrome, light A marked influence of NO fumigation was also
signaling, and plastid division and differentiation pro- observed on the mRNA abundance of tomato genes
teins was evaluated. This analysis revealed that, except encoding key components of the plastid division

Figure 3. NO fumigation promotes cotyledon

greening in tomato phytochromobilin-deficient
mutant seedlings. au seedlings dark grown for
120 h were transferred to continuous RL under
constant fumigation with NO-free air (control)
or different NO concentrations. A, Chlorophylls
and carotenoids. B, Plastid structure in au cot-
yledon cells after 72 h of treatment. Values
shown are means 6 SE. Asterisks indicate sta-
tistically significant differences compared with
the untreated control at each sampling time
(Student’s t test, P , 0.05). DW, Dry weight.

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Melo et al.

significantly higher transcript abundance in au than in

wild-type seedlings during most of the RL treatment
period (Fig. 4B; Supplemental Fig. S6). As observed for
the photomorphogenesis-related genes, NO fumigation
triggered the reduction in plastid division genes mRNA
in au seedlings toward similar, or even lower, amounts
than those observed in wild-type seedlings. Finally, it is
also worth mentioning that, upon NO fumigation, au
seedlings displayed a progressive increase in mRNA
amounts of SlGLK1, which encodes a key transcription
factor responsible for chloroplast differentiation in tomato
vegetative tissues (Fig. 4B; Supplemental Fig. S6). There-
fore, NO-induced chloroplast differentiation correlates
with the accumulation of SlGLK1 transcripts and a con-
comitant reduction in mRNA abundance of genes en-
coding key components of the plastid division machinery.

NO and Ethylene Antagonistically Interact during

Light-Driven Cotyledon Greening

Given the importance of ethylene during plant re-

sponses to light (Rodrigues et al., 2014; Van de Poel
et al., 2015; Weller et al., 2015) and its close interplay
with NO during diverse plant responses (Manjunatha
et al., 2012; Freschi, 2013), we next analyzed whether
NO-ethylene interactions regulate tomato seedling
greening and chloroplast development.
Whereas the endogenous levels of the ethylene pre-
Figure 4. NO fumigation restores wild-type transcript levels of photo-
cursor 1-aminocyclopropane-1-carboxylic acid (ACC)
morphogenesis-related genes in phytochromobilin-deficient mutant remained relatively stable in dark-grown wild-type and
seedlings maintained under RL. Wild-type (WT) and au seedlings dark au seedlings, a drastic reduction in endogenous ACC in
grown for 120 h were transferred to continuous RL under constant fu- wild-type seedlings was observed soon after exposure
migation with NO-free air or 150 mL L21 NO (au + NO). A, Quantitative to either RL or BL (Fig. 5A). In contrast, BL, but not RL,
reverse transcription-PCR analysis of genes encoding phytochromes triggered significant reductions in the ACC content of
and light signaling proteins. B, Quantitative reverse transcription-PCR au seedlings. The activity of ACC oxidase (ACO), a key
analysis of genes encoding proteins involved in plastid division and enzyme in ethylene production, and ethylene emission
differentiation. Values shown are means 6 SE. The mean relative ex- rates were significantly higher in the au mutant than in
pression was calculated from means of two technical replicates of at
wild-type seedlings under either complete darkness or
least three biological replicates and normalized against the samples
from wild-type 0-h samples. Different letters indicate statistically sig-
RL conditions (Fig. 5, B and C). Interestingly, fumiga-
nificant differences among the genotypes and treatments at each sam- tion of au seedlings with NO under RL conditions
pling time (P , 0.05) according to a permutation test that lacks triggered a significant reduction in ACC content, ACO
assumptions of sample distribution (Pfaffl et al., 2002). activity, and ethylene emission (Fig. 5), denoting a re-
pressor influence of NO on ethylene biosynthesis dur-
ing tomato seedling deetiolation.
machinery (Fig. 5B; Supplemental Fig. S5). The tomato Additionally, the impact of NO fumigation on eth-
genome possesses at least two paralogs of FILAMENTOUS ylene signaling was estimated by determining the ac-
TEMPERATURE SENSITIVE Z (SlFtsZ1 and SlFtsZ2), tivity of the reporter protein GUS expressed under the
which encode tubulin-like GTPases that assemble into a control of the EBS ethylene-responsive promoter
ring-like structure at the plastid division site, thereby (Stepanova et al., 2007). An enhanced ethylene signaling
representing a central component of the division pro- output was observed in au seedlings maintained under
cess (Basak and Møller, 2013). Interacting with FtsZ either complete darkness or RL treatment (Fig. 5D). NO
proteins, a number of structural and regulatory elements, fumigation significantly reduced EBS promoter activity
such as ACCUMULATION AND REPLICATION OF in RL-treated au seedlings (Fig. 5D). Interestingly, such a
CHLOROPLASTS (ARC), CRUMPLED LEAF (CRL), NO-triggered reduction in ethylene signaling output
and Min proteins, facilitate the correct placement, as- does not seem associated with changes in the tissue
sembly, and stabilization of the plastid-dividing FtsZ sensitivity to this hormone, since simultaneous treat-
ring (Basak and Møller, 2013). All six tomato plastid ments with exogenous NO and ethylene rendered EBS
division-associated genes analyzed in this study (SlFTsZ1, activity levels similar to those observed in seedlings
SlFTsZ2, SlARC3, SlARC6, SlCRL, and SlMinD) exhibited treated with ethylene alone (Supplemental Fig. S7).
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 NO-Hormone Cross Talk during Light-Evoked Greening

Figure 5. Light and NO down-regulate ethylene metabolism and signaling output during tomato seedling greening. Seedlings
dark grown for 120 h were either kept in darkness (D) or transferred to continuous BL, RL, or RL and continuous fumigation with
150 mL L21 gaseous NO (RL + NO). A, ACC content. B, ACO activity. C, Ethylene emission. D, In vitro GUS activity assayed in
EBS::GUS seedlings carrying the synthetic ethylene-responsive promoter EBS fused to the GUS reporter gene. Values shown are
means 6 SE. Asterisks indicate statistically significant differences compared with the dark treatment (in A–C) or with the wild type
(WT; in D) at each sampling time (Student’s t test, P , 0.05). au + NO, au seedlings fumigated with 150 mL L21 NO; DW, Dry
weight.

Given that NO down-regulates ethylene produc- and pigment levels (Fig. 7) under either RL or BL
tion in deetiolating tomato seedlings, we further in- treatment.
vestigated the ethylene effect on NO production.
Treating wild-type seedlings with gaseous ethylene
or its precursor ACC resulted in a drastic reduction in NO Positively Interacts with Auxins during Light-Driven
endogenous NO release, almost entirely abolishing Cotyledon Greening
the RL- or BL-driven up-regulation of NO production
(Fig. 6A). Of particular note, both these hormonal NO-auxin cross talk has been associated with numer-
treatments significantly reduced cotyledon greening ous plant developmental responses (Freschi, 2013; Sanz
in deetiolating wild-type seedlings (Fig. 7A). Similar et al., 2015). In wild-type tomato seedlings, RL-evoked
analyses conducted in seedlings of the ethylene- deetiolation was accompanied by a 10-fold increase in the
insensitive Never ripe (Nr) mutant confirmed the re- endogenous indole acetic acid (IAA) content (Fig. 8, A
pressor influence of ethylene on NO production in and B). In contrast, RL-exposed au seedlings exhibited
deetiolating tomato seedlings. Compared with the a very modest increment in IAA endogenous levels,
wild type, Nr seedlings released higher amounts of which never exceeded 3 times the IAA levels observed
NO (Fig. 6A), which positively correlated with total under continuous darkness. Interestingly, NO fumi-
NR activity (Fig. 6B), NR activation state (Fig. 6C), gation of RL-treated au seedlings strongly promoted
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Melo et al.

Figure 6. Ethylene negatively influences endog-

enous NO production in deetiolating tomato
seedlings. Seedlings dark grown for 120 h were
transferred to continuous RL or BL treatment.
Treatment of wild-type (WT) seedlings with
100 mL L 21 gaseous ethylene (ET) or 100 mM ACC
was initiated 48 h before starting the light exposure.
Ethylene concentration was renewed daily, and
ACC was supplemented only once to the growth
medium. Seedlings of the ethylene-insensitive
Never ripe (Nr) mutant were not subjected to eth-
ylene or ACC treatment. A, Fluorometric quantifi-
cation of endogenous NO release. B, NR maximum
activity (NRmax). C, NR activation state. Values
shown are means 6 SE. Asterisks indicate statisti-
cally significant differences compared with the
wild type at each sampling time (Student’s t test,
P , 0.05). DW, Dry weight.

the accumulation of IAA in cotyledon tissues, which endogenous levels observed under these circumstances
exceeded IAA levels detected in RL-exposed wild-type (Fig. 8).
seedlings (Fig. 8B). The relative transcript amounts of The positive influence of NO on auxin content and
SlARF4, a member of the tomato auxin response factor signaling output prompted us to evaluate whether al-
gene family that acts as a repressor of the auxin re- terations in auxin signaling could also affect NO en-
sponse (Sagar et al., 2013), was markedly higher in au dogenous levels. To test this, endogenous NO was
than wild-type seedlings under RL conditions (Fig. 8C). measured in tomato deetiolating mutant or transgenic
SlARF4 mRNA abundance in au was reduced drasti- seedlings with altered auxin responsiveness. The auxin-
cally upon NO fumigation, achieving levels even lower insensitive tomato mutant diagetropica (dgt) exhibited
than those detected in RL-treated wild-type seedlings reduced NO levels when compared with wild-type
(Fig. 8C). Reinforcing a positive interaction between seedlings (Fig. 8, E and F). In contrast, RL- or BL-
NO, auxins, and light signaling, the auxin-responsive exposed seedlings of entire, a mutant overresponsive
promoter DR5 was strongly activated in au seedlings to auxins due to a single-base deletion in the coding
simultaneously treated with RL and NO fumigation region of the auxin signaling transcriptional repressor
(Fig. 8D). In the absence of NO fumigation, RL expo- AUX/IAA9 (Zhang et al., 2007), or an SlARF4-silenced
sure failed to stimulate the DR5 promoter in au, which line (Sagar et al., 2013) exhibited significantly higher
is consistent with the very modest increment in IAA endogenous NO release compared with wild-type
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
 NO-Hormone Cross Talk during Light-Evoked Greening

Figure 7. Ethylene inhibits chlorophyll and ca-

rotenoid accumulation in deetiolating tomato
seedlings. Photosynthetic pigment accumula-
tion is shown in cotyledons of wild-type (WT)
seedlings treated with gaseous ethylene (ET) or
ACC as well as in the ethylene-insensitive Nr
mutant exposed to either RL or BL treatment.
Treatment details are as described in Figure 6. A,
Chlorophylls. B, Carotenoids. Values shown are
means 6 SE. Asterisks indicate statistically sig-
nificant differences compared with the wild type
at each sampling time (Student’s t test, P , 0.05).
DW, Dry weight.

seedlings (Fig. 8, E and F). Collectively, these data sug- NR-Dependent NO Production Is Controlled by Light and
gest a mutually positive feedback between auxins and Plant Hormones
NO during tomato seedling deetiolation.
In plants, NR is arguably the NO-generating route
most clearly characterized at both the biochemical and
regulatory levels (Lea et al., 2004; Planchet and Kaiser,
DISCUSSION
2006b; Planchet et al., 2006). In agreement with previ-
The rapid acquisition of functional chloroplasts is of ous reports (Becker et al., 1992; Goud and Sharma, 1994),
obvious importance for seedlings exposed to light and tomato NR activity was shown to be phytochrome-
determines the transition of a purely heterotrophic me- dependently induced upon light exposure and re-
tabolism to an essentially autotrophic behavior (Waters pressed by HP1/DDB1 protein (Fig. 2). Interestingly, the
and Langdale, 2009). Not surprisingly, light is the central light-evoked increase in NO levels always coincided
environmental cue eliciting and orchestrating the endog- with significant increases in NR activity in both wild-
enous signaling events responsible for controlling chlo- type and mutant tomato seedlings, and all distinct
roplast differentiation (López-Juez et al., 2008); however, strategies employed to inhibit the induction of this
little is known about the cross talk between light and other enzyme successfully reduced NO production (Table I).
signaling pathways during this photomorphogenic re- Moreover, a strict temporal correlation between the
sponse. The free radical NO, for instance, has been dem- highest NR and NO levels was observed regardless of
onstrated to influence cotyledon and leaf greening when the light treatment applied (i.e. RL or BL; Fig. 2F;
applied exogenously (Beligni and Lamattina, 2000; Zhang Supplemental Fig. S4). Therefore, besides being the
et al., 2006; Liu et al., 2013), but the role of endogenous NO main NO-generating route involved in numerous
in light-evoked chloroplast differentiation and the mech- plant defense responses (for review, see Mur et al.,
anisms responsible for NO production and action during 2013), our data demonstrated that NR is also the major
this photomorphogenic event have remained fairly elu- source of NO during tomato seedling greening. In
sive so far. Here, we provide a line of evidence for the addition to the reported influence of auxins, cytoki-
involvement of NO in a complex regulatory network that nins, and ABA on NR gene expression and enzyme
interconnects light perception, auxins, and ethylene to- activity (Lu et al., 1992; Yu et al., 1998), our study
ward the acquisition of photomorphogenic traits in dee- demonstrated that ethylene negatively affects both NR
tiolating tomato seedlings. total activity and activation state (Fig. 6), revealing
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Melo et al.

Figure 8. NO and auxin positive interactions during tomato seedling greening. Seedlings dark grown for 120 h were either kept in
darkness or transferred to continuous BL, RL, or RL and continuous fumigation with 150 mL L21 gaseous NO (au + NO). A and B,
IAA endogenous levels. C, SlARF4 relative expression. D, In vitro GUS activity assayed in seedlings carrying the synthetic auxin-
responsive promoter DR5 fused to the GUS reporter protein (DR5::GUS). E and F, Fluorometric quantification of endogenous NO
release. Values shown are means 6 SE. Asterisks indicate statistically significant differences compared with the wild type (WT) at
each sampling time (Student’s t test, P , 0.05). DW, Dry weight; e, entire; SlARF4-ASL, SlARF4-silenced line.

that NR-dependent NO production in tomato seed- NO (Fig. 3), suggesting that NO might complement in
lings is under the strict control of both light and hor- some way the partial deficiency in the RL perception
mone signals. characteristic of this phytochromobilin-deficient mu-
tant. Etiolated au seedlings contain a small amount of
Endogenously Produced or Exogenously Applied NO functional phytochromes, which is estimated to range
Promotes Greening between 3% and 5% of wild-type levels (Parks et al.,
1987), thus conferring some residual RL perception and
In wild-type seedlings of grasses such as wheat response to this mutant. Therefore, the stimulatory
(Beligni and Lamattina, 2000) and barley (Hordeum effect of NO on seedling greening apparently depends
vulgare; Zhang et al., 2006; Liu et al., 2013; Chen et al., on the previous or concomitant activation of at least
2014), the positive influence of NO on seedling greening some phytochrome molecules, which agrees with
has been demonstrated by the increment in chlorophyll earlier observations about the importance of concom-
content upon the application of sodium nitroprusside itant light pulses for the NO-induced partial greening
(SNP), an NO donor. However, in wild-type tomato in etiolated wheat seedlings (Beligni and Lamattina,
seedlings, no alterations were observed after NO fu- 2000). In addition to these pharmacological data,
migation (Supplemental Fig. S4). This may reflect the analyses of mutant tomato seedlings with altered light
significant differences in the pharmacological ap- or hormone perception or signaling revealed that in-
proach employed in these studies, since SNP de- tensified greening always coincided with increased
composition generates not only NO but also other NO production (Figs. 2 and 6). Moreover, a clear
physiologically active compounds such as cyanide temporal coincidence was observed between the en-
(Bethke et al., 2006). Alternatively, this may also indicate dogenous NO generation and the greening process.
that endogenous NO production in light-exposed wild- NO and chlorophyll levels increased concomitantly
type tomato seedlings is already sufficient to trigger within the first 24 h of light exposure, whereas the
maximal photosynthetic pigment accumulation, thereby maximal accumulation of photosynthetic pigments
explaining the lack of further increases in chlorophyll and was clearly preceded by a transitory peak in en-
carotenoid levels upon NO fumigation (Supplemental dogenous NO levels (Fig. 2F; Supplemental Fig. S4).
Fig. S4). Therefore, the positive influence of NO on tomato
However, dose-response greening was observed in seedling greening is here supported by both genetic and
au seedlings simultaneously exposed to RL and gaseous pharmacological evidence.
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
 NO-Hormone Cross Talk during Light-Evoked Greening

NO Fumigation Regulates Photomorphogenesis-Related of both light and hormone signaling cascades (Halliday
and Plastid Division Genes and Fankhauser, 2003; Lau and Deng, 2010). According
to our data, the light-evoked greening in tomato seed-
Light perception is known to repress Arabidopsis lings is at least partially controlled by a mutual antag-
PHYA and PHYB gene expression during seedling dee- onism between NO and ethylene metabolisms. Several
tiolation (Somers and Quail, 1995). Consistent with these lines of evidence seem to support this finding. First,
findings, the transcript abundance of most tomato genes regardless of the genotype, the time course of NO and
encoding phytochrome apoproteins was significantly ethylene production was clearly opposite during light-
higher in au than in wild-type seedlings under RL, and triggered seedling deetiolation. Second, any experi-
such differences were consistently reverted upon NO mental condition resulting in increased NO production
fumigation (Fig. 4). These observations indicate that NO also caused significant reductions in ACC levels, ACO
can restore the feedback mechanisms responsible for activity, and ethylene emission. Third, NO fumigation
repressing phytochrome gene expression upon light ex- drastically reduced the enhanced ethylene biosynthesis
posure. Interestingly, it has also been proposed that NO and signaling observed in RL-treated au seedlings.
modulates PHYB protein levels in Arabidopsis seedlings Similar to the au mutant, increased ethylene production
under RL (Lozano-Juste and León, 2011). In addition, also has been demonstrated in pea (Pisum sativum)
SNP application was reported to influence PHYA mRNA phytochrome-deficient plants (Foo et al., 2006). Treat-
levels in etiolated wheat seedling leaves maintained un- ments with ethylene biosynthesis inhibitor or mutation in
der continuous darkness (Liu et al., 2013), suggesting that the ethylene signaling gene ETHYLENE INSENSITIVE2
NO might act as a light-independent upstream regulator rescued many of the phenotype alterations observed in
of phytochrome gene expression. In our system, the NO- this pea mutant (Foo et al., 2006; Weller et al., 2015).
imposed reduction in phytochrome apoprotein mRNA Therefore, light perception via phytochromes is believed
levels took place under RL conditions; therefore, it does to play a critical role in reducing ethylene synthesis to
not necessarily indicate a direct action of NO as an up- levels compatible with the induction and progression of
stream regulator of phytochrome gene expression, but key photomorphogenic responses (Vangronsveld et al.,
obviously such a possibility cannot be ruled out. Re- 1988; Foo et al., 2006; Weller et al., 2015).
gardless of acting upstream or downstream of phyto- The NO-ethylene antagonism detected in tomato
chromes, NO fumigation led to a progressive reduction seedlings seems to rely mainly on NO-triggered re-
in transcript levels of the photomorphogenic repressor striction of ethylene biosynthesis rather than on
proteins SlCOP1, SlCUL4, SlDET1, and SlDDB1, imply- changes in the tissue sensitivity to this hormone. This is
ing a gradual increase in tissue responsiveness to light evident by the reduction in ACC content and ACO ac-
stimuli (Jiao et al., 2007), which, in turn, could have fa- tivity followed by the significant decrease in ethylene
cilitated the conversion of the residual RL perception of emission observed in NO-fumigated tomato seedlings
au seedlings into photomorphogenic responses. (Fig. 5) and is in agreement with pharmacology-based
The influence of both light and NO on the plastid di- experiments that have suggested NO as a potent in-
vision machinery has received little attention in the liter- hibitor of ethylene biosynthesis during the onset of fruit
ature (Basak and Møller, 2013). Our data demonstrated ripening and leaf senescence (Leshem et al., 1998; Wills
that both of these signals can modulate the mRNA et al., 2000; Manjunatha et al., 2010, 2012). As NO an-
abundance of key plastid division genes (Fig. 4). The tagonizes ethylene biosynthesis and promotes seedling
impairment in phytochrome-dependent light perception greening, a negative impact of ethylene on the chloro-
in the au mutant induced the mRNA accumulation of phyll accumulation might be expected. In fact, deetio-
genes involved in plastid division, such as SlFTsZ2, lating wild-type seedlings treated with ACC or gaseous
SlARC3, SlARC6, SlCRL, and SlMinD (Fig. 4). This might ethylene exhibited impaired cotyledon greening and
be either a direct effect of the absence of phytochrome- reduced NO levels, while the ethylene-insensitive Nr
dependent signaling or the lack of a negative feedback mutant displayed significantly higher NO, chlorophyll,
regulation of the plastid division machinery by fully and carotenoid contents (Figs. 6 and 7).
mature chloroplasts (Basak and Møller, 2013). Upon NO Over the last 2 years, significant progress has been
fumigation, the mRNA level of plastid division genes de- made in our understanding of the mechanisms re-
creased at the same time that transcripts of the key chlo- sponsible for NO sensing and signal transduction in
roplast differentiation gene SlGLK1 accumulated (Fig. 4B; plants (Gibbs et al., 2014; Abbas et al., 2015). The basis
Supplemental Fig. S6). Hence, NO apparently represents for this progress was the identification of NO as a key
one of the endogenous signals responsible for coordinating signal controlling the degradation of group VII ethyl-
the balance between plastid biogenesis and differentiation ene response factors (VII-ERFs) by the N-end rule
in response to environmental light conditions. pathway of targeted proteolysis (Gibbs et al., 2014).
Interestingly, NR was shown to represent the main bio-
NO Interconnects Light and Plant Hormones during synthetic source of NO responsible for modulating the
Seedling Greening turnover of VII-ERFs during early Arabidopsis devel-
opment, and the abundance of these transcription factors
The conversion of light stimuli into photomorpho- seems critical for controlling key photomorphogenic
genic responses usually involves the integrated action events such as hypocotyl elongation (Gibbs et al., 2014),
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Melo et al.

apical hook opening, and cotyledon greening (Abbas accumulation and signaling but auxin signaling itself
et al., 2015). Supporting this view, it has been demon- also stimulated endogenous NO production. As evidence
strated that apical hook opening is repressed signifi- of this, seedlings with exaggerated auxin responsiveness,
cantly in the nia1nia2 NR-deficient double mutant, a such as the entire mutant or SlARF4-silenced lines,
phenotype clearly reverted upon NO fumigation or in a showed increased endogenous NO content, whereas the
combination mutant where VII-ERFs were knocked out opposite was observed in the auxin-resistant mutant dgt
in the NR-deficient mutant background (Abbas et al., (Fig. 8). This is consistent with the increased NO pro-
2015). Moreover, data obtained by Abbas et al. (2015) duction detected upon exogenous auxin application
also suggest that NO promotes Arabidopsis cotyledon (Pagnussat et al., 2002; Correa-Aragunde et al., 2004; Hu
greening by catalyzing the destruction of VII-ERFs, et al., 2005; Lombardo et al., 2006) or in Arabidopsis
since these transcription factors were shown to repress auxin overproducer mutants (Chen et al., 2010). Finally,
the expression of key chlorophyll biosynthesis genes. considering that auxins have been reported to regulate
These findings are consistent with our working hy- NR at both the transcriptional and posttranscriptional
pothesis that NR-derived NO promotes tomato coty- levels (Vuylsteker et al., 1997) and that the NR-deficient
ledon greening through a close interplay with ethylene Arabidopsis mutant fails to generate NO in response to
signaling and also suggest substantial conservation of exogenous auxin (Kolbert et al., 2008), it seems tempting
the mechanisms responsible for the NO-dependent to speculate that this enzyme is responsible for the auxin-
regulation of early photomorphogenic development triggered changes in NO production observed in this
between Arabidopsis and tomato. study.
Multiple cross talk possibilities between light and Although not explored in this study, another NO-
auxin transduction networks have been described in hormone interaction critically important for seedling
plants, including a light-driven impact on auxin bio- photomorphogenesis consists of the intricate interplay
synthesis, catabolism, transport, and tissue respon- between NO and gibberellins (GAs) (Lozano-Juste and
siveness (for review, see Halliday et al., 2009), but their
impacts on seedling greening have remained largely
unexplored. Our data point out that, unlike ethylene,
NO and auxins interact positively during tomato
seedling deetiolation. A marked increase in IAA con-
tent was induced in wild-type tomato seedlings upon
illumination, which was accompanied by a reduction in
the mRNA accumulation of the repressor of auxin re-
sponse SlARF4 and, consequently, the activation of the
auxin-responsive promoter DR5. These changes were
observed in RL-treated au seedlings exclusively upon
NO fumigation (Fig. 8). Therefore, as observed in some
other plant species (Symons and Reid, 2003; Halliday
et al., 2009), phytochrome-dependent light perception
promotes auxin accumulation and signaling during
tomato seedling deetiolation. Moreover, our data also
indicate that NO can modulate auxin metabolism and/
or transport, and thereby auxin signaling, in tomato
cotyledons. This NO-auxin interaction has been repor-
ted in other systems and physiological events (Xu et al.,
2010; Fernández-Marcos et al., 2011; Terrile et al., 2012).
Although the exact mechanisms through which NO
and auxin interact in plants still deserve further eluci-
dation, the fact that NO has been shown to promote
auxin-dependent gene expression via posttranslational Figure 9. Diagram illustrating light, NO, ethylene (ET), and auxin (AUX)
modifications of the Arabidopsis auxin receptor protein cross talk during the acquisition of photomorphogenic traits in tomato
TRANSPORT INHIBITOR RESPONSE1 is of particular seedlings. During light-evoked cotyledon greening and chloroplast
note (Terrile et al., 2012). This finding implies that differentiation, negative and positive feedback regulatory loops or-
changes in auxin tissue sensitivity may be modulated chestrate NO-ET and NO-AUX interactions, respectively. The mutual
by NO; therefore, the activation of auxin signaling upon NO-ET antagonism occurs at biosynthetic levels involving the negative
NO fumigation in tomato seedlings might reflect not regulation of NR and ACO by ET and NO, respectively. By repressing
negative regulators of light signal transduction such as DET1, DDB1,
only the increased auxin levels detected under these and COP1 and modulating the AUX-ET balance, NO promotes the
circumstances (Fig. 8) but also some increment in auxin accumulation of the chloroplast differentiation transcription factor GLK
tissue responsiveness. and inhibits key plastid division genes such as FTsZs, ARCs, CRL, and
Our data exposed a mutually positive feedback reg- Min. Biosynthetic enzymes are represented with gray ovals. Arrows at
ulatory loop during NO-auxin cross talk in tomato the ends of lines indicate stimulatory effects, whereas bars indicate
cotyledons, because not only did NO promote auxin inhibitory effects.

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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
 NO-Hormone Cross Talk during Light-Evoked Greening

León, 2011; Freschi, 2013). A mounting body of evi- modified, as appropriate. In the nitrogen-free MS medium, NH4NO3 was
omitted and KNO3 was replaced by 0.35 g L21 KCl and 0.4 g L21 K2SO4.
dence has indicated that a mutual NO-GA antagonism Modified MS medium containing ammonium or Gln as the exclusive source of
regulates hypocotyl elongation in Arabidopsis mainly nitrogen was essentially the same formulation as the nitrogen-free MS medium
due to the positive influence of NO on the accumulation with the addition of 1.61 g L21 NH4Cl or 2.19 g L21 Gln, respectively. Therefore,
of DELLA proteins (Lozano-Juste and León, 2011). In when present in the medium, nitrogen was always available at a final con-
agreement, a dramatic reduction in GA1 levels was centration of 30 mM. The medium containing Gln was sterilized by ultrafiltra-
tion, whereas the others were autoclaved.
reported in pea seedlings soon after transfer from Hormonal and NO treatments were initiated 72 h before starting the light
darkness to light (Symons and Reid, 2003; Weller et al., treatments and were maintained thereafter. ACC was dissolved in water,
2009), which would imply increased DELLA levels sterilized by ultrafiltration, and applied directly to the growth medium
upon light exposure. Interestingly, DELLAs are con- containing the seedlings. Treatments with gaseous ethylene (100 mL L21) and
NO (50, 100, or 150 mL L21) were conducted in sealed magenta boxes (total
sidered to act as promoters of greening (Cheminant airspace volume of 360 mL). Ethylene application was renewed on a daily
et al., 2011); therefore, the NO-GA antagonism already basis. NO fumigation was performed by mixing a commercial standard
described during the regulation of hypocotyl elonga- mixture (10 mL L21 NO in nitrogen) with NO-free breathing air at appro-
tion (Lozano-Juste and León, 2011) may also be in- priate flow rate ratios. NO gas mixtures were bubbled through a flask con-
volved in the light-evoked cotyledon-greening process. taining 500 mM KOH to eliminate any nitrous acid traces and humidify the
gas mixture. A continuous flow (100 mL min21) was maintained throughout
Further studies focusing on NO-GA interactions during the experiments, and final NO concentration was routinely checked by
cotyledon greening are clearly required to substantiate injecting 1-mL aliquots of each magenta box’s internal atmosphere into a
this possibility. chemiluminescence detector (CLD 88ep; Eco-Physics), as described by Fre-
In conclusion, this study brings evidence of a close schi et al. (2010).
interaction between NO, ethylene, and auxins during
the light-induced etioplast-to-chloroplast conversion in Chlorophyll and Carotenoid Quantification
deetiolating tomato seedlings. During this process, the
For chlorophyll and carotenoid extraction, samples of cotyledons were
light-induced production of NO via NR activity ap- weighed (typically 20 mg fresh weight), immersed in a 1003 excess volume of N,
parently antagonizes ethylene production and inten- N-dimethylformamide, and incubated for 48 h at 25°C in absolute darkness.
sifies auxin accumulation in cotyledon tissues. As a The supernatant absorbance was recorded at 480, 647, and 664 nm, and the total
consequence, negative and positive feedback regula- chlorophyll and carotenoid contents were estimated using the equations pub-
lished by Porra et al. (1989) and Wellburn (1994).
tory loops apparently orchestrate ethylene-NO and
auxin-NO interactions, respectively (Fig. 9). Therefore,
NO seems to represent an important element inter- NO Measurements
connecting phytochrome-dependent light perception, For fluorometric NO determination, the cell-impermeant fluorophore DAR-
auxins, and ethylene toward the acquisition of fully 4M was used. Cotyledons were gently fragmented into small pieces (typically
mature chloroplasts in deetiolating seedlings. 5 3 5 mm), immediately weighed (approximately 100 mg fresh weight), and
incubated with 1 mL of 50 mM phosphate buffer (pH 7.2) containing 37.5 mM
DAR-4M for 30 min at 25°C in absolute darkness on a rotary shaker (200 rpm).
The supernatant fluorescence was measured using a spectrofluorometer
MATERIALS AND METHODS (LS55; Perkin Elmer) with 560-nm excitation and 575-nm emission wavelengths
(5-nm bandwidth). Fluorescence was measured at the same instrument settings
Plant Material and Growth Conditions in all experiments and was expressed as arbitrary fluorescence units per gram
dry weight per hour.
Wild-type tomato (Solanum lycopersicum ‘Micro-Tom’) and the near-isogenic
lines harboring the mutations au, hp1, entire, dgt, and Nr were obtained by
Carvalho et al. (2011). The wild type, near-isogenic lines, and transgenic cv NR Activity Assay and Activation State
Micro-Tom carrying the synthetic auxin-responsive (DR5) and ethylene-
NR maximum activity and activation state were assayed according to Tucker
responsive (EBS) promoters fused to the reporter gene uid (encoding a GUS)
et al. (2004) with some modifications. Briefly, samples were ground in liquid
were obtained from the tomato mutant collection maintained at the Escola
nitrogen and subsequently homogenized in extraction buffer (approximately
Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo (http://
300 mg tissue fresh weight mL21) composed of 100 mM HEPES-KOH (pH 7.6),
www.esalq.usp.br/tomato/). Seeds of the cv Micro-Tom SlARF4-silenced line
10 mM FAD, 10 mM Na2MoO4, 1 mM EDTA, 0.5% (w/v) polyvinylpyrrolidone,
(SlARF4-ASL) were obtained from the University of Toulouse (Sagar et al.,
5 mM dithiothreitol (DTT), and 10 mM leupeptin. After centrifugation
2013). Additionally, crosses and phenotypical screening were performed to
(13,000g, 10 min, and 4°C), 100 mL of the supernatant was added to 900 mL
generate the double mutants au-DR5::GUS and au-EBS::GUS.
of reaction medium composed of 100 mM HEPES-KOH (pH 7.6), 10 mM FAD,
5 mM DTT, 20 mM KNO3, 200 mM NADH, and 10 mM EDTA (for NR maximum
Growth Conditions and Treatments activity) or 15 mM MgCl2 (for actual NR activity). The reactions were incu-
bated for 5 min at 30°C and then stopped by adding 100 mL of 500 mM zinc
Seeds were surface sterilized with 0.5% (v/v) sodium hypochlorite and sown acetate. The unreacted NADH was oxidized by incubation with 100 mM
directly in magenta vessels containing sterile medium composed of one-half- phenazine methosulfate for 20 min, and the nitrite produced by the reactions
strength MS salts and 2% (w/v) Phytagel. After 120 h of pregermination in was quantified by standard colorimetric reaction (Man et al., 1999). For the
absolute darkness, seedlings were transferred to continuous RL or BL or determination of NR maximum activity, the same extract was preincubated in
maintained under absolute darkness. RL and BL were supplied by an array of ice for 12 min in the presence of 20 mM AMP, 15 mM EDTA, and 10 mM
SMD5050 Samsung light-emitting diodes mounted in a temperature-controlled KH2PO4. In all cases, blanks were made by adding zinc acetate to the reaction
growth chamber maintained at 25°C 6 1°C and installed inside a darkroom. medium prior to the addition of the protein extract. NR activation state is
Both BL and RL were delivered at approximately 50 mmol m22 s21, with peak given as the ratio of maximum NR activity (measured after preincubation of
output at 470 and 625 nm, respectively, as defined by the manufacturer. In all extracts with excess EDTA plus AMP) and the values of NR activity (mea-
cases, tissue was harvested either under the specific light conditions used for sured in the presence of excess Mg2+; Man et al., 1999). NR activities obtained
seedling growth or under dim green light, as appropriate. in the presence of excess Mg2+ are believed to reflect NR activity in situ,
For the treatment of seedlings with inhibitors of NR activity, seeds were whereas maximum NR activity indicates the total pool of NR protein avail-
grown in the same manner described above except that the MS medium was able in the tissues (Man et al., 1999).

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Melo et al.

Ethylene Measurements measuring the ability of the extract to convert exogenous ACC to ethylene after
incubation at 30°C for 20 min. The ethylene levels were measured by gas
Ethylene emission was analyzed as described by Freschi et al. (2010). Briefly, chromatography-flame ionization detection, as described above.
intact tomato seedlings (typically 100 individuals) growing inside sealed ma-
genta boxes were flushed with ethylene-free air (1 L min21) for 5 min and in-
cubated for 24 h under specific experimental conditions, as appropriate. After Transmission Electron Microscopy
incubation, 1-mL gas samples were withdrawn with a gas-tight syringe and
Cotyledons were cut into small pieces (1 3 1 mm) and fixed with 2.5% (w/v)
injected into a Trace Ultra gas chromatograph (Thermo Electron) fitted with a
glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2), postfixed in 1% (w/v)
flame ionization detector and an RT-Alumina Plot column (Restek). Nitrogen
osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2), dehydrated in a
was used as the carrier gas at a flow rate of 3 mL min21, and commercial
graded acetone series, and embedded in Spurr’s resin. Semithin sections were
standard mixtures of ethylene were used for the calibration curves. Column,
stained with 1% (w/v) Toluidine Blue, and the ultrathin sections were counter-
injector, and detector temperatures were 40°C, 250°C, and 250°C, respectively.
stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and
analyzed with a Zeiss EM 900 transmission electron microscope. The electron
IAA and ACC Measurements micrographs illustrate the relevant results obtained.

Endogenous IAA and ACC levels were determined simultaneously by gas

chromatography-tandem mass spectrometry-selective ion monitoring. Ap- Quantitative PCR
proximately 200 mg fresh weight of frozen cotyledon samples was ground in
RNA extraction and quantitative PCR (qPCR) were performed as described
liquid nitrogen in the presence of 20 mg of polyvinylpyrrolidone and subse-
by Lira et al. (2014) with some modifications. Total RNA was extracted from
quently homogenized in 1 mL of extraction solution composed of 10 mM
200 mg fresh weight of powdered cotyledon material using the Plant RNeasy
ascorbic acid, 10 mM EDTA, and 10 mM DTT. Exactly 1 mg of [13C6]IAA
Kit (Qiagen) and subsequently treated with the TURBO DNA-free Kit (Ambion).
(Cambridge Isotopes) and 2 mg of [2H4]ACC (Sigma-Aldrich) were added to
The complementary DNA was synthesized using the SuperScript III One-Step RT-
each sample as internal standards. After centrifugation (25,000g, 20 min, and
PCR System (Invitrogen), as recommended by the manufacturer. mRNA levels
4°C), the supernatant was purified via solid-phase extraction (Supelclean
were quantified using a 7500 Real-Time PCR system (Applied Biosystems) and
LC-NH2 column; Supelco), as described by Purgatto et al. (2002). Purified
SYBR Select Master Mix (Applied Biosystems). Data were analyzed as described
samples were evaporated, resuspended in 50 mL of pyridine, followed by a 60-
by Lira et al. (2014). All reactions were performed with two technical replicates
min derivatization at 92°C using 50 mL of N-tert-butyldimethylsilyl-N-methyl-
and four biological replicates. Expression values were normalized against the
trifluoroacetamide. The analysis was performed on a gas chromatograph coupled
geometric mean of two reference genes, SlUBIQ and SlGAGA. All primers used
to a mass spectrometer (model GCMS-QP2010 SE; Shimadzu) in selective ion
are shown in Supplemental Table S1.
monitoring mode. The chromatograph was equipped with a fused-silica capillary
column (30 m, 0.25 mm i.d., and 0.25-mm-thick internal film) DB-5 MS stationary
phase using helium as the carrier gas at a flow rate of 4.5 mL min–1 in the Statistical Analysis
following program: 2 min at 100°C, followed by a ramp of 10°C min –1 to
140°C, 25°C min–1 to 160°C, 35°C min–1 to 250°C, 20°C min–1 to 270°C, and Statistical analyses were performed using the Jump statistical software
30°C min–1 to 300°C. The injector temperature was 250°C, and the following package (14th edition) and Microsoft Excel 2010. Test results with P # 0.05 were
mass spectrometry operating parameters were used: ionization voltage, 70 eV considered statistically significant.
(electron-impact ionization); ion source temperature, 230°C; and interface
temperature, 260°C. Ions with a mass ratio/charge of 244, 202, and 130 (cor- Sequence data from this article can be found in the GenBank/EMBL data
responding to endogenous IAA), 250, 208, and 136 (corresponding to [13C6] libraries under accession numbers.
IAA), 272, 244, and 188 (corresponding to endogenous ACC), and 276, 248,
and 192 (corresponding to [2H4]ACC) were monitored. Endogenous concentra-
tions were calculated based on extracted chromatograms at mass ratio/charge
244 and 250 for IAA and 272 and 276 for ACC. Supplemental Data
The following supplemental materials are available.
Quantitative GUS Activity Assay
Supplemental Figure S1. Plastid structure in wild-type and mutant tomato
GUS activity was assayed according to Jefferson et al. (1987) with some seedlings exposed to RL, BL, and white light.
modifications. Briefly, cotyledon samples were ground in liquid nitrogen and
Supplemental Figure S2. Detailed time course of greening, NO levels, and
subsequently homogenized in 4-methylumbelliferyl-b-D-glucuronide ex-
NR activity in wild-type seedlings.
traction buffer (approximately 150 mg tissue fresh weight mL–1) composed of
50 mM HEPES-KOH (pH 7), 5 mM DTT, and 0.5% (w/v) polyvinylpyrrolidone. Supplemental Figure S3. Influence of pharmacological treatments with
After centrifugation (13,000g, 20 min, and 4°C), 200-mL aliquots of the super- NOS-like inhibitor.
natant were mixed with 200 mL of GUS assay buffer composed of 50 mM
Supplemental Figure S4. NO fumigation effects under continuous dark-
HEPES-KOH (pH 7), 5 mM DTT, 10 mM EDTA, and 2 mM 4-methylumbelliferyl-
ness or BL.
b-D-glucuronide and incubated at 37°C for 30 min. Subsequently, aliquots of
100 mL were taken from each tube, the reactions were stopped with 2.9 mL of Supplemental Figure S5. qPCR data of genes encoding phytochromes and
0.2 M Na2CO3 (pH 9.5), and fluorescence was determined using a spectrofluo- light signaling proteins.
rometer (LS55; Perkin Elmer) with 365-nm excitation and 460-nm emission
wavelengths (5-nm bandwidth). Fluorescence was measured at the same instru- Supplemental Figure S6. qPCR data of genes encoding plastid division
ment settings in all experiments. and differentiation proteins.
Supplemental Figure S7. Tissue sensitivity to ethylene in NO-treated seed-
lings.
Measurement of ACO Activity
Supplemental Table S1. Primers used for qPCR.
The extraction and activity assay of ACO was carried out according to
Vriezen et al. (1999) with some modifications. Briefly, samples were ground in
liquid nitrogen and subsequently homogenized in extraction buffer (approxi-
mately 500 mg tissue fresh weight mL–1) composed of 300 mM Tris-HCl (pH 8), ACKNOWLEDGMENTS
50 mg mL–1 insoluble polyvinylpyrrolidone, 10% (v/v) glycerol, and 30 mM
We thank Mondher Bouzayen (University of Toulouse) for providing seeds
sodium ascorbate. After centrifugation (20,000g, 20 min, and 4°C), 200 mL of the
of the cv Micro-Tom SlARF4 antisense line.
supernatant was added to 1.7 mL of reaction medium composed of 100 mM Tris-
HCl (pH 8), 10% (v/v) glycerol, 30 mM sodium ascorbate, 100 mM FeSO4, 50 mM Received January 13, 2016; accepted January 29, 2016; published February 1,
NaHCO3, 5 mM DTT, and 2 mM ACC. ACO activity was determined by 2016.

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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
 NO-Hormone Cross Talk during Light-Evoked Greening

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