The diversity of nitric oxide function in plant responsesto metal stress

Huyi He • Longfei He • Minghua Gu

Received: 20 January 2014 / Accepted: 28 January 2014

� Springer Science+Business Media New York 2014

Abstract Nitric oxide (NO) emerges as signalling

molecule, which is involved in diverse physiological

processes in plants. High mobility metal interferes

with NO signaling. The exogenous NO alleviates

metal stress, whereas endogenous NO contributes to

metal toxicity in plants. Owing to different cellular

localization and concentration, NO may act as mul-

tifunctional regulator in plant responses to metal

stress. It not only plays a crucial role in the regulation

of gene expression, but serves as a long-distance

signal. Through tight modulation of redox signaling,

the integration among NO, reactive oxygen species

and stress-related hormones in plants determines

whether plants stimulate death pathway or activate

survival signaling.

Keywords Nitric oxide � Metal stress �Reactive oxygen species � Hormones �Cross talk � Programmed cell death

Abbreviations

ABA Abscisic acid

ACO 1-Aminocyclopropane-1-carboxylate

oxidase

ACS 1-Aminocyclopropane-1-carboxylate

synthase

APX Ascorbate peroxidase

As Arsenic

BRs Brassinosteroids

CAT Catalase

Cu Copper

Cd Cadmium

cPTIO 2-(4-Carboxyphenyl)-4,4,5,5-

tetramethylimidazoline-1-oxyl-3-oxide

CTK Cytokinin

DAF-FM 4-Amino-5-methylamino-20,70-difluorofluorescein

ET Ethylene

GSNOR1 S-Nitrosoglutathione reductase gene

Hsp71.2 Heat shock protein 71.2

JA Jasmonic acid

L-NMMA NG-Monomethyl-arginine monoacetate

MAPK Mitogen-activated protein kinase

MAT-1 Met adenosyltransferases

Ni Nickel

NO Nitric oxide

NOS Nitric oxide synthase

NOX NADPH oxidase

NR Nitrate reductase

PA Polyamines

H. He (&) � L. He � M. Gu

College of Agronomy, Guangxi University,

Nanning 530004, People’s Republic of China

e-mail: wingtiger2008@hotmail.com

H. He

Cash Crops Research Institute, Guangxi Academy

of Agricultural Sciences, Nanning 530007,

People’s Republic of China

123

Biometals

DOI 10.1007/s10534-014-9711-1

PAL Phenylalanine ammonialyase

PCD Programmed cell death

POX Peroxidase

PrP4A Pathogen-related proteins

ROS Reactive oxygen species

SAG12 Senescence-associated gene 12

SNP Sodium nitroprusside

Zn Zinc

ZR Zeatin

Introduction

Water, air, and plants are contaminated by metals,

which result from environmental deterioration. With

the acceleration of industrialization process and wide

use of phosphate fertilizers, metals are transferred to

the food chain and present a potential threat to

human health (Jarup and Akesson 2009). Metal

toxicity has become one of the major abiotic stress

agents leading to hazardous health effects in animals

and plants. For plants, high reactivity metals not only

are taken up by roots, but also can be translocated in

aerial organs. Through disruption of water and

nutrient uptake, inhibition of photosynthesis and

nitrogen metabolism, metal stress results in growth

inhibition, structure damage, a decline of physiolog-

ical and biochemical activities of plants. The effects

of metal stress on different plant species may be

opposite. For example, cadmium (Cd) stimulates

mRNA coding for phenylalanine ammonialyase

(PAL) in soybean, but caused a decrease of PAL

mRNA level in lupine (Pawlak-Sprada et al. 2011).

The effects and bioavailability of metals depend on

environmental conditions, pH, species of element,

organic substances of the media and fertilization, and

plant species. The metals combination by proteins,

exudation of organic acids, and expression of detox-

ifying enzyme are integral to protect the plants

against injury by metal stress. Understanding the

bioavailability of metals is advantageous for plant

cultivation and phytoremediation.

Nitric oxide (NO) is an important diffusible

signalling molecule, which participates in a variety

of physiological processes in plants, including seed

germination, adventitious root formation, pro-

grammed cell death (PCD), flowering, stomatal clo-

sure and defence responses (Besson-Bard et al. 2008).

The mechanisms of NO signal transduction include

cGMP-dependent signaling pathway and cADPR-

dependent free cytosolic Ca2? signaling (Wilson

et al. 2008). NO also can regulate the expression of

target proteins by post-translational modification. In

animal cells, NO is synthesized from L-arginine

oxidation through heme-containing nitric oxide syn-

thase (NOS). However, the sources of NO synthesis in

plants are complex and have not yet been resolved

(Neill et al. 2002). Besides nitrate reductase (NR)-

dependent and nonenzymatic NO production, a puta-

tive NOS-like enzyme-dependent reaction has been

highlighted in recent years (Crawford 2006). During

the adventitious rooting process, NO and cGMP are

involved in the auxin response. A mitogen-activated

protein kinase (MAPK) signaling cascade is also

activated in a NO-mediated pathway (Pagnussat et al.

2004). However, there is little detailed information on

the sources of NO in plants, its functions in plants as

well as on the mechanisms underlying its effects.

Metals treatment alters NO contents in plants. NO also

has been demonstrated to be a key modulator in the

resistance response in plant against metals such as

Cd2? (Groppa et al. 2008; Laspina et al. 2005; Singh

et al. 2008; Xiong et al. 2009a), Cu (Singh et al. 2004;

Yu et al. 2005; Hu et al. 2007; Zhang et al. 2008;

Tewari et al. 2008; Zhou et al. 2012), Ni (Mihailovic

and Drazic 2011; Kazemi 2012), Zn (Abdel-Kader

2007), and As (Singh et al. 2009). But the reports on

the effects of exogenous and endogenous NO in metal

stress are conflicting. In this review, we focus on the

latest advances in understanding the effects of metals

on endogenous NO content and the role of exogenous

NO in metal stress in plants. On the basis of crosstalk

between NO, reactive oxygen species (ROS) and

stress-related hormones, a mode of NO action under

metal stress is also proposed.

Metal stress and NO content in plants

Cd treatment for 72 h is accompanied by a rapid NO

increase in Arabidopsis cell suspension cultures (De

Michele et al. 2009). Treatment with 200 lM Cd for

7 h induces a strong increase of NO in the roots of

Arabidopsis thaliana, while a treatment of 50 lM Cd

for 96 h also induces an increase of NO in the leaves of

Arabidopsis (Besson-Bard et al. 2009). The induction

of NO generation by treatment with 1 mM Cd for 24 h

Biometals

123

was observed in pericycle, parenchymatic stelar cells

and companion cells of protophloem of barley roots

(Valentovicova et al. 2010). As evidenced by the

fluorescent probe 4-amino-5-methylamino-20,70-diflu-

orofluorescein (DAF-FM), NO content was found to

be significantly increased in the roots of Cd treated

plants. During 5 days, Cd-induced NO formation in

wheat roots was directly correlated with root growth

inhibition (Groppa et al. 2008). A dose-dependent and

rapid production of NO was exhibited in soybean cells

treated with 4 or 7 lM Cd for 72 h (Kopyra et al.

2006). NO increases in the roots of Brassica Juncea L.

and Pisum sativum L. exposed to 100 lM Cd for

7 days. When treated with 0.1 mM CdCl2 for 5 days,

NO content in the root apices of the wheat seedlings

increases (Corpas et al. 2008). Long-term treatment

with 1 lM Cd for 4 weeks produces a 2.4-fold

increase in NO and short-term (3 h) treatment with

10 lM Cd induces a 73 % increase of NO content in

wheat roots (Mahmood et al. 2009). A fast NO burst in

the first 6 h was followed by a slower, gradual increase

with Cu load (Bartha et al. 2005). With the duration

and concentration of Cu exposure, NO accumulates in

Chalmydomonas reinhardtii (Zhang et al. 2008). NO

increases in the adventitious roots of Panax ginseng

exposed to 50 lM Cu for 24 h (Tewari et al. 2008).

Exposure to Cu induced NO production in the root tips

of Indian mustard and rapeseed (Feigl et al. 2012).

Especially, Cu and Cd treatments resulted in 2- to 3.2-

fold enhancement of NO production in white poplar

cell cultures, while NO production were not changed

in the zinc-treated cells (Balestrazzi et al. 2009). Low

concentration Zn induced leaf structural modifications

to maintain functional integrity, may be a compensa-

tory strategy to enhance Zn tolerance (Di Baccio et al.

2013). Lead (Pb) triggered NO burst in root cells of

Pogonatherum crinitum by enhancing NR activity (Yu

et al. 2012).

In contrast, Treatment with 100 lM Cd for 24 h

significantly decreases the NO content in the crown

roots of 7-day-old rice seedlings (Xiong et al. 2009a).

NO in the roots of Medicago truncatula significantly

decreases after treatment with 50 lM Cd for 48 h (Xu

et al. 2010a). Treatment with 50 lM Cd for 14 days also

produces a significant reduction of NO content in the

roots and leaves of P. sativum (Rodriguez-Serrano et al.

2009; Barroso et al. 2006; Rodriguez-Serrano et al.

2006). Metal stress can promote NO production or

inhibit NO accumulation. The discrepancy is a

consequence of different metal concentrations, the

species and/or form of metal, the age of plants, the

duration or treatment and the variety of plant tissues

used. Certainly, the different plant growth systems,

sampling and detection techniques are also potential

causes.

Role of NO in metal stress in plants

The contribution of endogenous NO to plant metal

stress

As a cell-signalling molecule, NO exerts cytotoxic or

protective effects depending on its surrounding micro-

environment and its sources. The mammalian NOS

inhibitors L-NAME and PBITU suppressed Cd2?-

induced NO production, which was not suppressed in

the double mutant nia1 nia2, suggesting NO might be

catalyzed through a L-Arg dependent pathway rather

than a nitrate/nitrite dependent route. Al toxicity leads

to the reduction of endogenous NO concentration,

which is required for root elongation growth in plant

(He et al. 2012).

Moreover, it has been proposed that NO plays a role

in the regulation of iron homeostasis and in the plant

responses to toxic metals. NO can inhibit the activity

of aconitase by affecting Fe–S group (Navarre et al.

2000). Through metal nitrosylation of protein Fe, NO

is a key component of responsive mechanism in plant

Fe metabolism, whether iron deficiency (Graziano and

Lamattina 2007) or excess Fe stress (Arnaud et al.

2006). By promoting Cd2? versus Ca2? uptake, NO

favours Cd2? accumulation in roots and contributes to

root growth inhibition by partly preventing the Cd2?-

induced repression of the Fe-starvation responsive

genes IRT1, FRO2 and FER-LIKE FEDEFICIENCY-

INDUCED TRANSCRIPTION FACTOR 1 (FIT1)

(Besson-Bard and Wendehenne 2009). Owing to

diffusible or responsive features, NO is produced not

only locally, but also in systemic tissues. By inducing

GENERAL REGULATORY FACTOR 11 (GRF11)-

dependent FIT expression, NO mediates Fe deficiency

responses (Yang et al. 2013).

The recent reports that NO contributes to Cd-

induced cell death in plants are listed (Table 1). By

modulating Cd2? uptake and thus promoting Cd2?

accumulation in tobacco BY-2 cells, NO played a

positive role in CdCl2-induced PCD consistent with

Biometals

123

the increase of Hsr203J expression (Ma et al. 2010). A

relatively early burst of NO localized mainly in root

tips precedes Cd-induced PCD (Arasimowicz-Jelonek

et al. 2012). NO promotes Cd2?-induced Arabidopsis

PCD by promoting MPK6-mediated caspase-like

activation (Ye et al. 2013). Just as the increase of

senescence-associated gene 12 (SAG12) expression

showed, NO is involved in Cd-induced PCD in

Arabidopsis cell suspensions by modulating the con-

centration and function of PC through protein S-nit-

rosylation (De Michele et al. 2009). NO regulates the

activity of metacaspase by S-nitrosylation of cysteine

residue (Belenghi et al. 2007). Caspase-like proteases

are involved in Cd-induced cell death in tomato

suspension cells (Iakimova et al. 2008). 50–100 lM

CdSO4 induced apoptotic-like PCD, while 1,000 lM

Cd showed strong cytotoxicity with DNA fragmenta-

tion (Fojtova and Kovarik 2000). NO contributes to Cd

toxicity by promoting Cd accumulation in roots and

up-regulating genes related to iron uptake (Besson-

Bard and Wendehenne 2009). Cd-induced NO gener-

ation functions in Cd toxicity through the ectopic and

accelerated differentiation of barley root tips, causing

the shortening of the root elongation zone and a

subsequent reduction in root growth (Valentovicova

et al. 2010). Exogenously applied NO inhibits root

growth to a similar extent as Cd does (Groppa et al.

2008). Under the condition of Cd-depressed NOS-

dependent NO production, the pathogen-related pro-

teins (PrP4A), chitinase, and the heat shock protein

71.2 (Hsp71.2) were up-regulated (Rodriguez-Serrano

et al. 2009). NO promotes Cd uptake and subsequent

metal-induced reduction of root growth. NO contrib-

utes to Cd toxicity in Arabidopsis thaliana by medi-

ating an iron deprivation response, which opens new

windows in the understanding of NO function in metal

toxicity (Besson-Bard and Wendehenne 2009). Cd

decreases crown root number by decreasing endoge-

nous NO, which is indispensable for crown root

primordia initiation in rice seedlings (Xiong et al.

2009a). The white poplar cultures exposed to metals

showed the morphological hallmarks of both PCD and

necrosis, which were associated with the increase of

NO production (Balestrazzi et al. 2009). The interplays

between NO and ROS promoted Zn-induced PCD in

Solanum nigrum root tips, subsequently modulated

root system architecture to adapt Zn toxicity (Xu et al.

2010b). NO promotes Cd stress by reducing the

expression and activity of S-nitrosoglutathione reduc-

tase gene (GSNOR) in pea plants significantly (Barroso

et al. 2006). Overexpression of rice GSNOR1 allevi-

ated H2O2-induced leaf cell death in nitric oxide

excess1 (noe1) rice (Lin et al. 2012). NO has both

promoting and suppressing effects on cell death,

depending on many factors such as cell type, cellular

redox status, and the flux and dose of local NO.

Exogenous NO alleviates metal stress in plants

As an antioxidant, pretreatment with an NO donor

scavenges ROS and enhances tolerance to metal stress

in plants. NO reduces Cd-induced phytotoxicity in

Table 1 Reports on NO contribution to metal-induced cell death in plants

Plant species Metals NO-mediated effect References

Triticum aestivum Cd Inhibits root growth Groppa et al. (2008)

Oryza sativa Cd Initiates crown root primordia Xiong et al. (2009a)

Oryza sativa Cd Variation in the levels of NPT,

PBT, and matrix polysaccharides

Zhang et al. (2012)

Arabidopsis thaliana Cd Increases ROS production De Michele et al. (2009)

Arabidopsis thaliana Cd Promotes Cd2? accumulation Besson-Bard et al. (2009)

Arabidopsis thaliana Cd Mediates an iron deprivation response Besson-Bard et al. (2009)

Arabidopsis thaliana Cd Promotes MPK6-mediated caspase-3-like activation Ye et al. (2013)

Arabidopsis thaliana Fe Induces protein degradation Arnaud et al. (2006)

Hordeum vulgare Cd Accelerates differentiation of root tips Valentovicova et al. (2010)

Nicotiana tabacum Cd Modulates Cd influx Ma et al. (2010)

Lupinus luteus Cd Enhances post-stress signals level Arasimowicz-Jelonek et al. (2012)

Solanum nigrum Zn Induces PCD Xu et al. (2010b)

Populus alba Cu, Zn, Cd Induces PCD and necrosis Balestrazzi et al. (2009)

Biometals

123

wheat roots (Groppa et al. 2008). Cd toxicity is

reduced by NO in rice leaves (Hsu and Kao 2004). NO

protects sunflower leaves against Cd-induced oxida-

tive stress (Laspina et al. 2005). A NO donor, sodium

nitroprusside (SNP) supplementation ameliorates Cd

toxicity in hydroponically grown wheat roots through

prevention of oxidative stress (Singh et al. 2008).

Treatment of plants with artificially generated NO

protect plant tissues against the oxidative damage

triggered by Cd by promoting the scavenging of ROS

directly through chemical processes or indirectly via

the activation of ROS-scavenging enzymes (Kopyra

et al. 2006; Noriega et al. 2007). By preventing

oxidative stress, exogenous NO alleviates Cd toxicity

of Lipinus luteus (Kopyra and Gwozdz 2003). Exog-

enous NO enhances Cd tolerance of rice by increasing

pectin and hemicelluloses contents in root cell wall

and decreasing Cd accumulation in soluble fraction of

shoot (Xiong et al. 2009b). Exogenous NO recovers

Cd-induced crown root primordia initiation in rice

seedlings (Xiong et al. 2009a). Exogenous NO

improves antioxidative capacity, reduces auxin deg-

radation and enhances ion absorption in roots of M.

truncatula seedlings under Cd stress (Xu et al. 2010a).

Exogenous application of NO improved the various

morpho-physiological and photosynthetic parameters

in control as well as Cd-treated plants (Jhanji et al.

2012). Associated with elevated NO levels, CaCl2mitigated the growth inhibition of CdCl2 on rice

seedlings significantly (Zhang et al. 2012). SNP

delayed Cd-promoted flowering in Arabidopsis, which

was associated with the increase of NO accumulation

in leaves (Wang et al. 2012). Exogenous NO can

effectively facilitate structural adjustment in P. sati-

vum leaves under Cd stress, which could improve

stress tolerance at the whole-plant level (Tran et al.

2012). Nevertheless, it was reported that SNP pre-

treatment can promote ROS-mediated Cd cytotoxicity

in B. juncea (Verma et al. 2013). As an integral

modulator, NO/H2O2 can ameliorate Cd or Cu-

induced toxicity in Scenedesmus quadricauda (Stork

et al. 2013). The addition of SNP in combination with

Cu to N-NH4?-grown Chlorella significantly reduced

the oxidative burst (Singh et al. 2004). The reduction

of Cu2?-induced toxicity and NH4? accumulation by

SNP is most likely mediated through its ability to

scavenge ROS (Yu et al. 2005). Pretreatment with

SNP could significantly improve wheat seeds germi-

nation and alleviate oxidative stress against Cu

toxicity (Hu et al. 2007). The endogenous NO

generated was positively associated with the proline

level in Cu-stressed algae. Pre-treatment of SNP

increased the proline accumulation in Cu-treated cells

by about 1.5-fold (Zhang et al. 2008). Exogenous NO

improved the activities of SOD and NADPH oxidase

in excess Cu supplied adventitious roots of mountain

ginseng (Tewari et al. 2008). Application of SNP

efficiently alleviated the Cu toxicity effects in root tips

of Vicia faba L. (Zhou et al. 2012). Exogenous NO

efficiently attenuates oxidative stress in bean, but does

not prevent Ni-induced ion leakage (Mihailovic and

Drazic 2011). NO sequestration by Ni in the roots

increased antioxidant enzyme activity and markedly

reduced Ni-induced oxidative damage on tomato

plants (Kazemi 2012). SNP maintains a suitable zinc

concentration in both wheat and bean seedlings, which

may be a result of the adjustment of free/total SH

levels, glutathione content and SOD activity (Abdel-

Kader 2007). By partially reversing arsenic (As)-

induced oxidative stress, exogenous NO provides

resistance to rice against As-toxicity (Singh et al.

2009). SNP and S-nitrosoglutathione (GSNO) can

reduce Pb accumulation in P. crinitum root cells (Yu

et al. 2012). Such studies provide an alternative route

for crops improvement under metal stress, but this

route also have some limits, namely, the amelioration

effects depend on the concentration of exogenous NO

used in the experiments. Moreover, high concentration

of exogenous NO even enhances metal toxicity in

plants.

Exogenously applied NO can alleviate metal tox-

icity in plants, promoting the direct scavenging of

ROS or activating antioxidant enzymes. However, NO

even contributes to Cd toxicity by promoting Cd

uptake and participates in Cd-induced reduction of

root growth. Certainly, in order to generate a mobile

distal signal, active cell death is required for enhanced

tolerance responses in neighboring cells or other upper

plant organs (Overmyer et al. 2003).

Cross-talk among NO, ROS and stress-related

hormones

NO potentiates ROI-induced hypersensitive cell death

in soybean cells, so it functions as a signal in disease

resistance in plants (Delledonne et al. 1998; De

Stefano et al. 2005). Accompanying by the NADPH

Biometals

123

oxidase (NOX)-dependent superoxide anion (O2�-)

production, roots of 3-day old yellow lupine seedlings

exposed to Cd resulted in a relatively early burst of

NO. Boosted NO and O2�- production is required for

Cd-induced PCD in lupine roots (Arasimowicz-Je-

lonek et al. 2012). S-nitrosylation of peroxiredoxin II

E promotes peroxynitrite-mediated tyrosine nitration

(Romero-Puertas et al. 2007). It is reversibly that NO

is able to inhibit the activities of catalase (CAT) and

ascorbate peroxidase (APX) in tobacco hypersensitive

response (Clark et al. 2000). De Michele et al. (2009)

showed inhibition of NO synthesis by NG-mono-

mehyl-arginine monoacetate (L-NMMA) resulted in

partial prevention of H2O2 increase under Cd stress.

NO/ROS interaction may be cytotoxic or protective

depending on the relative balance of redox signaling

(Beligni and Lamattina 1999). NO can S-nitrosylate

GAPDH, which cysteine residues are oxidized by

H2O2, suggesting GAPDH may be intersection

between regulatory pathways of two signaling mole-

cules (Lindemayr et al. 2005). Through protein

S-nitrosylation, NO may regulate the activity of

NtOSAK containing S-nitrosylated characteristic

domain and promote the production of cytosolic

Ca2? (Lamotte et al. 2006). With considerable cross-

talk between responses to several stimuli, how NO

cooperates with ROS to trigger PCD in intact plants

exposed to Cd is still far from being clarified.

Besides the role of the hormones in plant response

to Cd, the interactions NO with pathways of phyto-

hormones also are involved in Cd stress signaling

(Table 2). Obtained from linolenic acid, jasmonic acid

(JA) production is associated with lipid peroxidation

and membrane damage. Through the activation of

lipoxygenase activity, H2O2 production, and lipid

peroxidation, the increase of JA could contribute to Cd

toxicity (Rodriguez-Serrano et al. 2009). The expres-

sion of JA-induced defence genes are affected by NO

donors (Grun et al. 2006). The results of biotin switch

proteomics showed that S-nitrosylation of allene oxide

cyclase (AOC) may be an important link between NO

and JA biosynthesis (Zeigler et al. 2000). NO could

inhibit 1-aminocyclopropane-1-carboxylate synthase

(ACS) or 1-aminocyclopropane-1-carboxylate oxi-

dase (ACO) to prevent ET formation, so ethylene

(ET) and NO are antagonistic (Leshem 2000). The Cd-

dependent reduction of the NO level in leaves could

alter Met adenosyltransferases (MAT-1) regulation by

S-nitrosylation and increase ET biosynthesis (Rodri-

guez-Serrano et al. 2009). The salicylic acid (SA)

positive feedback loop is essential for amplifying the

distal signal in the upper zone of the plant, so SA is

engaged in plant response to Cd (Guo et al. 2009). The

application of the NO-scavenger 2-(4-carboxy-

phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-

oxide (cPTIO) during Cd stress lowered the SA

synthesis in lupine leaves (Arasimowicz-Jelonek

et al. 2012). An interrelationship between NO and

SA is involved in plant defence, because the accumu-

lation and function of SA was impaired in AtGSNOR1

deficiency Arabidopsis mutants (Feechan et al. 2005).

Gibberellic acid (GA) remarkably reduced NO accu-

mulation, which partially be reversed by the applica-

tion of NO donor S-nitrosoglutathione (GSNO) (Zhu

Table 2 Reports on relationship between NO and stress-related hormones

Plant species Stress-related

hormones

The species

of metal

Level References

Pisum sativum JA Cd ? Rodriguez-Serrano et al. (2009)

ET Cd ? Rodriguez-Serrano et al. (2009)

Lupinus luteus SA Cd ? Arasimowicz-Jelonek et al. (2012)

Arabidopsis thaliana Auxin Cu - Peto et al. (2011)

GA Cd - Zhu et al. (2012)

ZR Cd - Vitti et al. (2013)

Oryza sativa ABA Cd ? Hsu and Kao (2005)

Medicago truncatula IAA Cd ? Xu et al. (2010a)

Triticum durum CTK Cd - Veselov et al. (2003)

Triticum aestivum BR Cd ? Bajauz (2011), Kroutil et al. (2010)

PA Cd ? Groppa et al. (2008)

Biometals

123

et al. 2012). With the increase in dose and duration of

Cd exposure in Myriophyllum heterophyllum and

Potamogeton cripus, the content of abscisic acid

(ABA) increased (Sivaci et al. 2008). ABA accumu-

lation helps to enhance Cd tolerance in rice seedlings

(Hsu and Kao 2005). NO may participate in main-

taining the IAA equilibrium by reducing IAA oxidase

activity in roots of M. truncatula subjected to Cd

stress, thus alleviating the negative effect of Cd on root

growth inhibition (Xu et al. 2010a). NO intensifies Cu-

induced cotyledon expansion, but mitigates elongation

processes. Auxin and NO negatively regulate each

other’s level under Cu2? exposure (Peto et al. 2011).

Cd Supplementation for 2 h sharply reduced cytokinin

(CTK) content in wheat seedlings by elevating CTK

oxidase activity (Veselov et al. 2003). A shorter root

axis length and the doubled diameter of their lateral

roots were showed in Cd-treated Arabidopsis seed-

lings, accompanying by significant changes in the

levels of IAA, trans-ZR riboside, dihydrozeatin ribo-

side (DHZR) (Vitti et al. 2013). Brassinolide (BL)

enhanced the content of IAA, zeatin (ZR), and ABA in

cultures treated with Cd, suggesting BL plays the

positive role in the alleviation of Cd stress (Bajauz

2011). The treatment of spring wheat with brassinos-

teroids (BRs) decreased Cd content in the growth stage

73–75 DC (30–50 % of final grain size) (Kroutil et al.

2010). Treatment of wheat roots with either Cd or

polyamines (PA), especially those exposed to sperm-

ine, gave a common response, illustrated in an

enhanced NO formation that mediated toxicity exerted

by these compounds and resulted in root growth

inhibition (Groppa et al. 2008). Metal possibly induces

signaling pathways, especially those connected with

jasmonate, ethylene and H2O2. In the case of longer

exposure to metal stress, the integration of different

signal transduction pathways ultimately lead to a fast

decrease of growth processes or accelerated senes-

cence in plants.

Conclusions and future perspectives

Being a diffusible signaling molecule, NO plays an

important role in the regulation of cell responses to

metals. A hypothetical model depicting NO in

response to metals is presented in Fig. 1. Metal stress

alters NO content in different cell compartment and

triggers ROS production. ROS-causing oxidative

damage could be partially responsible for death

pathway. In turn, NO indirectly triggers cellular

defense responses via sensors (including Ca2? fluxes,

cGMP, redox status, and MAPK cascades). Moreover,

NO also regulates the expression of post-stress genes,

such as Hsr203J, SAG12, GSNOR, Chitinases, PrP4A,

HSP71.2, PAL, peroxidase (POX), etc. (De Michele

et al. 2009; Rodriguez-Serrano et al. 2009; Barroso

et al. 2006; Ma et al. 2010; Hsu and Kao 2005). But at

the protein level the regulation still lack relevant

research data. Subsequently, the cross-talk between

NO, ROS and hormone equilibrium are involved in the

regulation of plant defense against metals, determin-

ing whether plants stimulate death pathway or activate

survival signaling.

In summary, some findings about role of NO in

metal stress contradict each other. The more we learn

about NO, the more its Janus face becomes apparent.

Metals may induce an increase or a decrease in NO

synthesis, which will result in dual action of metals—

induce both passive and active dying out of plant cells.

On the one hand, NO enhances metal accumulation

and induces active cell death. On the other hand, as a

distal signal, NO is implicated in promoting survival

signaling towards tolerance in plants. The controver-

sial relationship between NO and metal stress are

passive

active

Metal stress

ET /auxinSA/JA/PA

ROS

Death pathway Survival signaling

NO

Gene expression

sensors

Fig. 1 The hypothetical mode of NO action in plant responses

to metal stress. Metal-dependent changes of NO and ROS

contents can be perceived by sensors (Ca2? fluxes, cGMP, redox

status, and MAPK cascades). The transcriptional response can

also require more upstream signal transduction involving

sensors and hormones such as JA, auxin, SA, PA and ET.

Finally, the integration between NO, ROS and stress-related

hormones in plants determines cell fate. Whether plants

stimulate death pathway or activate survival signaling depends

on different plant species, concentration and duration of metals

treatment. Among death pathway, one is gene-controlled active

program—PCD. Another is ROS-mediated passive process. The

arrows indicate activation. Blocked lines indicate repression

Biometals

123

attributed to the influences of metals on NO content

and the different sources of NO generation in plants.

Perhaps the application of NO donors does not truly

reflect endogenous NO signaling in plants. To monitor

the NO content in plants, we have to develop better

experimental systems and assay method. What is the

interrelationship between cGMP-dependent signal

pathway and NO-involved protein modification?

How to achieve interactive regulation between NO-

involved protein modification and ROS components?

How to collaborate between protein ubiquitination and

S-nitrosylation in cell death? In addition, the identi-

fication of more NO targets will be a charming

challenge in future research. Decrease in the bioavail-

ability to farmlands would reduce the accumulation of

metals in food. Alternatively, one could increase the

bioavailability of plants to extract more metals.

Understanding the networks involved in plant

defenses against metal stress and the roles of NO in

regulating redox signaling might provide targets for

enhancing crop production.

Acknowledgments This work was supported by the National

Natural Science Foundation of China (No. 30960181 and

31260296) and 2011 Guangxi Innovation Program for

Graduates (GXU11T31076).

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