I g C C a b P c a A R R A K A A A B B D D E G G G J R R S S S 1 n a ( o r ( t a e 0 h Environmental and Experimental Botany 94 (2013) 73– 88 Contents lists available at SciVerse ScienceDirect Environmental and Experimental Botany jou rn al h om epa ge: www.elsev ier .com/ locate /envexpbot nteractions between hormone and redox signalling pathways in the control of rowth and cross tolerance to stress arlos G. Bartoli a, Claudia A. Casalonguéb, Marcela Simontacchia, Belen Marquez-Garciac, hristine H. Foyerc,∗ Instituto de Fisiología Vegetal, Facultad Ciencias Agrarias y Forestales, Universidad Nacional de La Plata-CCT CONICET La Plata, cc327 (1900) La Plata, Argentina Instituto de Investigaciones Biológicas, UE-CONICET-UNMDP, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250, cc1245 (7600) Mar del lata, Argentina Centre of Plant Sciences, Research Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK r t i c l e i n f o rticle history: eceived 31 January 2012 eceived in revised form 8 May 2012 ccepted 9 May 2012 eywords: uxin bscisic acid scorbate iotic stress rassinosteroids ELLA proteins rought thylene ibberellic acid lutathione a b s t r a c t The ability of plants to respond to a wide range of environmental stresses is highly flexible and finely balanced through the interaction of hormonal plant growth regulators and the redox signalling hub, which integrates information from the environment and cellular metabolism/physiology. Plant hormones produce reactive oxygen species (ROS) as second messengers in signalling cascades that convey infor- mation concerning changes in hormone concentrations and/or sensitivity to mediate a whole range of adaptive responses. Cellular redox buffering capacity that is determined largely by the abundance of ascorbate has a profound influence on the threshold at which hormone signalling is triggered and on the interactions between different hormones. Other antioxidants such as glutathione, glutaredoxins and thioredoxins are also central redox regulators of hormone signalling pathways. The complex network of cross-communication between oxidants and antioxidants in the redox signalling hub and the different hormone signalling pathways maximises productivity under stress-free situations and regulates plant growth, development, reproduction, programmed cell death and survival upon exposure to stress. This interactive network confers enormous regulatory potential because it allows plants to adapt to changing and often challenging conditions, while preventing boom or bust scenarios with regard to resources, ensuring that energy is produced and utilised in a safe and efficient manner. rowth asmonic acid eactive oxygen species edox signalling alicylic acid tress signalling hub © 2012 Elsevier B.V. All rights reserved. trigolactones . Introduction Cross-tolerance to environmental stresses is a common phe- omenon in plants, whereby exposure to one type of stress confers general increase in resistance to a range of different stresses Pastori and Foyer, 2002; Suzuki et al., 2012). Cross-tolerance ccurs because of synergistic co-activation of non-specific stress- esponsive pathways that cross biotic–abiotic stress boundaries Bostock, 2005). Cross-tolerance phenomena are frequently linked o the enhanced production of reactive oxygen species (ROS) such s H2O2, oxidative signalling and the associated regulation of gene xpression through the redox signalling hub, as illustrated in Fig. 1. ∗ Corresponding author. E-mail address: c.foyer@leeds.ac.uk (C.H. Foyer). 098-8472/$ – see front matter © 2012 Elsevier B.V. All rights reserved. ttp://dx.doi.org/10.1016/j.envexpbot.2012.05.003 It is widely accepted that H2O2 and other ROS are important signalling molecules in abiotic and biotic stress responses, often because they serve as messengers for the activation of defence genes (Foyer and Noctor, 2009, 2012). Tight spatial-temporal con- trol of redox signalling molecules allows different and sometimes diametrically opposed physiological events and generates signal specificity that is integrated with the action of plant hormones such as ethylene (ET), salicylic acid (SA), abscisic acid (ABA) and jasmonates (JA) (Xiong et al., 2002; Glazebrook et al., 2003; Fujita et al., 2006). For example, exposure to the atmospheric pollutant ozone generates ROS in the apoplast of plant cells and initiates an oxidative signalling cascade that shares many signalling and regulatory response components with ROS-mediated responses to biotic and abiotic stresses (Baier et al., 2005). Many plant hormones promote ROS production, often through the activation of NADPH oxidases (Rbohs), or alter redox signalling hormones and so induce dx.doi.org/10.1016/j.envexpbot.2012.05.003 http://www.sciencedirect.com/science/journal/00988472 http://www.elsevier.com/locate/envexpbot mailto:c.foyer@leeds.ac.uk dx.doi.org/10.1016/j.envexpbot.2012.05.003 74 C.G. Bartoli et al. / Environmental and Experimental Botany 94 (2013) 73– 88 ub. Re t A d r h d ( t t p a e c p 2 h o r m b e a 2 t d m t p a Fig. 1. The redox signalling h olerance to a wide spectrum of stresses (Foyer and Noctor, 2009). gain using the ozone example, ozone fumigation induces ET pro- uction (Nakajima et al., 2002) and the activation of abiotic stress esponses (Leubner-Metzger et al., 1998), as well as triggering the ypersensitive response (HR) and the induction of programmed cell eath, which are intrinsic feature of plant responses to pathogens Overmeyer et al., 2003). Extensive cross talk is observed between he SA, JA and ET pathways that are induced in response to oxida- ive stresses such as ozone (Glazebrook et al., 2003) with many oints of reciprocal control that can involve neutral, synergistic nd antagonistic interactions (Tosti et al., 2006). We discuss the vidence showing that the major plant hormones interact with the ellular redox signalling hub in order to control growth and defence rocesses in response to environmental stresses. . Auxin Auxin, principally indole-3-acetic acid (IAA) is an essential plant ormone that fulfils numerous roles in plant growth and devel- pment including stem elongation, phototropic and gravitropic esponses, apical dominance, and lateral and adventitious root for- ation (Grieneisen et al., 2007; Kleine-Vehn et al., 2008). Like rassinosteroids (BRs) and gibberellins (GAs), auxin promotes cell longation and controls plant height. BRs and auxin produce ROS s second messengers by activation of NADPH oxidases (Joo et al., 001; Xia et al., 2009, 2011). Plant development is regulated by precisely controlled fluc- uations in auxin biosynthesis, transport, accumulation, and egradation. The fine-tuning auxin concentrations with local auxin axima, directional cell-to-cell transport and auxin gradients, ogether with the differential distribution of the auxin signalling athways in specific tissues at specific stages of development llow the correct setting of developmental cues in embryogenesis, active oxygen species (ROS). organogenesis, vascular tissue formation and directional growth in response to environmental stimuli. Auxin transport is controlled by a family of influx facilitators (AUX1, LAX1–LAX3) and two fam- ilies PIN-FORMED (PIN) and type B ATP binding cassette proteins of efflux carriers. Much attention has focussed on the coordina- tion of the polar sub-cellular localisation of PIN proteins that are responsible for the direction of auxin fluxes (Friml, 2010). The auxin model is considered to be a paradigm for cellular signal transduc- tion pathways (Teale et al., 2008). Moreover, the dynamic interplay between auxin signalling pathways and redox signalling pathways (Fig. 2) permits flexible regulation that is highly responsive of cell metabolism (Pasternak et al., 2005; Tognetti et al., 2012). The balance between oxidative (ROS) and reductive (antiox- idant) signals regulates auxin biology at multiple levels from biosynthesis, conjugation/oxidation, and transport to signal trans- duction (Gazarian et al., 1998; Cosio and Dunand, 2009; Chen and Xiong, 2009; Tognetti et al., 2010). For example, ROS function as downstream components in auxin-mediated signal transduction to control gravitropism responses in roots (Joo et al., 2001). Genes encoding antioxidant enzymes are among primary auxin-response genes, suggesting a role for auxin in plant stress and defence responses (Abel and Theologis, 1996; Tyburski et al., 2009; George et al., 2010). Changes in auxin distribution and ROS metabolism precede transcriptional regulation (Joo et al., 2001; Pasternak et al., 2005). To date, relatively little information is available on the complex interplay between ROS and auxin signalling. However, there is evi- dence in support of a dynamic dialogue and reciprocal dependence between redox (ROS-antioxidant) signalling and the prioritisation of polar auxin transport and signalling. Polar auxin transport, which enables cells to establish local auxin concentrations, is a key fea- ture of auxin homeostasis that is actively controlled by PIN auxin efflux carrier family. The expression of at least some of the PIN C.G. Bartoli et al. / Environmental and Exp Fig. 2. A simple representation of the auxin signalling pathway, showing possible s o t K a t “ b m T e e b d e 3 R 2 t p t 2 s t t ( i l b e s t t p b P o r m ites of regulation by GSH, Grx and the cytosolic throredoxin h (Trxh). Reactive xygen species (ROS). ransporters is regulated by glutathione (Bashandy et al., 2010; oprivova et al., 2010). The PIN proteins are constantly cycled in nd out of the plasma membranes. They are exchanged between he membrane and the “early” endosomes by a process called constitutive cycling” that allows rapid changes in plasma mem- rane composition by virtue of the presence of a pool of plasma embrane proteins that are available in nearby early endosomes. he internalisation of PIN proteins occurs by a clathrin-dependent ndocytosis mechanism (Chen et al., 1998; Friml et al., 2002). Any xogenous or endogenous stimulus that perturbs cellular redox alance can activate auxin homeostasis because NADPH oxidase- ependent ROS production influences polar auxin transport (Joo t al., 2005). The activation of phosphatidylinositol 3-kinase (PtdIns -kinase), which produces PtdIns(3)P, is required for auxin-induced OS production by NADPH oxidases in the root cells (Joo et al., 005). PtdIns(3)P plays a regulatory role in endocytosis and vesicle rafficking in plants. Therefore, ROS and phospholipid signalling athways may cooperate in the control of PIN-dependent auxin ransport (Matsuoka et al., 1995; Kim et al., 2001; Zegzouti et al., 006). Flavonoids may also participate in this regulation via repres- ion of polar auxin transporters (Peer and Murphy, 2007) because hey are versatile modulators of PIN interactions with regula- ory proteins particularly, PP2AA phosphatase and PINOID kinase Brown et al., 2001; Santelia et al., 2008; Friml, 2010). A hypothetical model, in which the activation of the phospho- nositide signalling pathways and plasma membrane endocytosis eads to NADPH oxidase-mediated ROS production, was described y Leshem et al. (2007) in relation to the acquisition of salt tol- rance. ROS accumulate in different sub-cellular compartments uch as chloroplast and mitochondria in response to environmen- al stresses. Differential ROS accumulation at various sites within he cell might also depend on vesicle trafficking, according to the hosphorylation state of the PIN proteins that regulates membrane inding. Auxin signalling by the auxin receptor AUXIN-BINDING ROTEIN 1 (ABP1) inhibits the clathrin-mediated internalisation f PIN proteins. Thus, ABP1 acts as a positive regulator of clathrin ecruitment to the plasma membrane (Robert et al., 2010). While any uncertainties remain concerning the extent of cross-talk erimental Botany 94 (2013) 73– 88 75 between auxin-mediated vesicle trafficking, and the redox and phosphoinositide signal pathways, redox signals exert a major influence on auxin synthesis and auxin-vesicle transport (Meyer et al., in press). 2.1. The central role of glutathione and thioredoxins Glutathione (GSH), glutaredoxins (Grx), peroxiredoxins (Prxs), thioredoxins (Trxs) and NADPH-thioredoxin reductases (NTRs) are central elements of the thiol-disulphide redox regulatory hub of plant cells. These components are key regulators for many stress signalling pathways and responses (Gómez et al., 2004; Meyer et al., in press; Noctor et al., 2012). Glutathione can modulate the activ- ities of MAP kinases in a number of ways such as thiol-disulphide exchange and glutathionylation (Foyer and Noctor, 2005) and by influencing cytosolic Ca2+ signalling (Foreman et al., 2003; Evans et al., 2005). Little is known about the redox regulation of auxin-related proteins, or the extent to which they undergo reg- ulatory posttranslational modifications, such as glutathionylation and nitrosylation. Extensive cross talk exists between the reduced GSH/Grx sys- tem and the cytosolic Trx and in the control of auxin synthesis, transport and meristem development. Auxin synthesis and polar transport are perturbed in Arabidopsis mutants lacking the monoth- iol glutaredoxin (Grx) Atgrx17. The Atgrx17 mutants accumulate higher ROS levels than the wild type and they have defective cell cycle control at high temperature (Chen et al., 2011). Moreover, high temperature-induced membrane leakage was increased in the Atgrx17 mutants indicating that glutathione-mediated redox regu- lation is critical for auxin transport during heat stress (Chen et al., 2011). Knock out Arabidopsis mutants in the GSH1 gene, which encodes �-glutamyl cysteine synthetase (�-ECS) the enzyme that catalyses the first step of GSH biosynthesis pathway, are lethal at the embryo stage (Cairns et al., 2006). However, the rootmeristemless1 (rml1) mutant, which has a less severe mutation in the GSH1 gene and thus allows accumulation of about 5% of the wild-type glutathione contents, shows a striking phenotype because it fails to develop a root apical meristem while the shoot meristem is largely unaffected (Vernoux et al., 2000). When the rml1 mutant was crossed with mutants that are deficient in the two cytosolic NTRs (ntra, ntrb), the triple mutants that are deficient in both reduced Trx and GSH had an additive shoot meristemless phenotype (Reichheld et al., 2007). The cytosolic Trx, Trxh3 may therefore provide an alternative to the GSH/Grx reduction system for the regulation of shoot auxin metabolism and transport (Meyer et al., in press). The A. thaliana cad2 mutant, which has a less severe mutation in the GSH1 gene than the rml1 mutant, has about 25% of wild- type GSH contents and it is characterised by increased sensitivity to cadmium. The cad2 mutant produces less lateral roots than the wild type and has impaired auxin transport but it does not display a pin phenotype. Triple mutants deficient in ntra, ntrb and cad2 lack apical dominance and have a pin1 phenotype, without flower formation (Bashandy et al., 2010). The triple mutants show a limited ability to transport auxin (Bashandy et al., 2010). In addition to genetic approaches, the specific inhibitor of �- ECS, buthionine sulphoximine (BSO) has frequently been used to study the effects of glutathione depletion. Treatment of the tips of primary roots with BSO alters auxin transport (Koprivova et al., 2010). However, the auxin-resistant axr1 and axr3 mutants are less sensitive to BSO than the wild-type A. thaliana plants (Koprivova et al., 2010). GSH synthesis is also required for pollen germination and pollen tube growth (Zechmann et al., 2011). In this study, glu- tathione depletion was shown to result from disturbances in auxin metabolism and transport (Zechmann et al., 2011). 7 d Exp M a o t e i w i 2 i e m 2 u H e 2 t a g W I a S b ( a f e k t v a t m i a i a s y T p p a u 2 a R a t W e 2 a b 2 a p t 6 C.G. Bartoli et al. / Environmental an Once auxins reach the site of action, they activate a APK cascade, which modulates gene expression and represses uxin-dependent signalling (Kovtun et al., 2000). Biotic and abi- tic stresses also activate MAPK signalling pathways that trigger he expression of antioxidant and defence genes (Hirt, 2000). For xample, the expression of a cytosolic ascorbate peroxidase (APX1) s increased by high light stress through a MAPK-dependent path- ay (Davletova et al., 2005). Knock out mutants that are deficient n APX1 (apx1) have higher H2O2 levels (Davletova et al., 2005). .2. Auxin and stress signalling Auxin is considered to be a component of the plant stress- nduced morphogenic response (SIMR; Potters et al., 2007; Potters t al., 2009) that serves to limit the adverse effects of environ- ental stress (Pasternak et al., 2005; Park, 2007; Tognetti et al., 012). Many auxin responsive genes are repressed by abiotic stim- li such as wounding, oxidative stress and selenium (Pfeiffer and oftberger, 2001; Cheong et al., 2002; Joo et al., 2005; Van Hoewyk t al., 2008; Jain and Khurana, 2009; Huang et al., 2010; Kieffer et al., 010). The suppression of auxin signalling is considered to enhance olerance to biotic and abiotic stresses, with a reciprocity between uxin-dependent reprogramming of gene expression and that trig- ered in response to stress (Navarro et al., 2006; Park et al., 2007; olters and Jurgens, 2009; Wang et al., 2010). Auxin functions by directly binding to the TRANSPORT NHIBITOR RESPONSE1 (TIR1), the F-box protein subunit of n ubiquitin protein ligase (E3) called SCFTIR1, which controls CF-mediated targeted protein degradation. Auxin binding desta- ilises interactions between TIR1/AUXIN-BINDING F-BOX PROTEIN TIR1/AFB) families of auxin receptors controlling the expression of uxin-regulated genes. In the absence of auxin, the auxin response actors (ARFs, which are transcription factors that regulate the xpression of target genes) are bound to negative regulators that eep them in an inactive state. The binding of IAA to TIR1 liberates he ARFs allowing the expression of target genes. For example, acti- ation of the TIR/AFBs pathway by asymmetric auxin flow occurs t the root tip in response to a gravitropic stimulus and this leads o curvature of the roots (Pan et al., 2009). Arabidopsis tir1/afbs utants have higher levels of ascorbic acid and higher APX activ- ties implicating the TIR/AFBs signalling pathway in the control of ntioxidant metabolism (Iglesias et al., 2010). In addition to glutathione, glutaredoxins and Trx, Prx are also mportant components of the cellular redox hub (Dietz, 2008). Prx re antioxidative enzymes, which have a broad range of substrate pecificity, eliminating H2O2, alkyl hydroperoxides and perox- nitrite through Grx- and Trx-based peroxide reductase activity. hey are found in many compartments of plant cells. As well as articipating in redox signalling they protect the nuclei (1CPrx), lastids (2CPrxA,2CPrxB, PrxQ, and PrxIIE), cytosol (PrxIIB, PrxIIC, nd PrxIID) and mitochondria (PrxIIF) from excessive oxidation nder stressful conditions (Romero-Puertas et al., 2007; Dietz, 008). PrxIIE mediates the cross talk between reactive nitrogen nd redox signalling pathways (Romero-Puertas et al., 2007). Like OS, NO regulates auxin-mediated signalling cascades and controls diverse set of auxin-mediated processes, including developmen- al and defence responses (Lamattina et al., 2003; Neill et al., 2003; endehenne et al., 2004; Delledonne, 2005; Correa-Aragunde t al., 2007; Besson-Bard et al., 2008; Palavan-Unsal and Arisan, 009; Yoshioka et al., 2009). NO can reduce the level of glutathione nd other antioxidants and also maintain the auxin equilibrium y reducing IAA oxidase activity in stressed tissues (Xu et al., 010). ROS and NO-signalling therefore interact in the regulation of uxin-mediated mechanisms. ROS, antioxidant and NO-signalling athways participate in the regulation of cell division, differentia- ion and the programmed cell death, partly through the regulation erimental Botany 94 (2013) 73– 88 of the expression of genes such as thylakoid APX (Corpas et al., 2001; Tarantino et al., 2005; de Pinto et al., 2006; Zago et al., 2006). 3. Salicylic acid Salicylic acid is a phenolic phytohormone (monohydroxyben- zoic acid) required in the signal transduction cascades that regulate plant defence mechanisms against biotic and abiotic stresses. It is particularly important in systemic acquired response (SAR), which is a broad-spectrum plant immune response involving profound transcriptional reprogramming (Cao et al., 1994; Dempsey et al., 1999; Vlot et al., 2009; Rivas-San Vicente and Plasencia, 2011). Ara- bidopsis mutants with differential endogenous SA contents have been very useful in resolving SA functions and deciphering SA- mediated signal transduction pathways. For example, mutants that are defective in CONSTITUTIVE EXPRESSION OF PR GENES5 (cpr5) have constitutively elevated SA levels (Clarke et al., 2000), whereas sid2 is defective in isochorismate synthase and hence can- not produce SA (Wildermuth et al., 2001), npr1 cannot respond to the SA signal because of a lesion in the NONEXPRESSOR OF PATHOGENESIS-RELATED genes 1 (NPR1) transcriptional regula- tor (Cao et al., 1994). Other constitutive defence mutants often have elevated SA levels and show growth retardation phenotypes (Robert-Seilaniantz et al., 2011). SA is synthesised either from phenylalanine via cinnamic acid or from chorismate by isochorismate synthase. SA acts as a central regulator of cell fate by the reprogramming of gene expression, a process that involves the activation of plasma membrane-bound NADPH oxidases. Together with cell wall peroxidases, NADPH oxi- dases are responsible for the oxidative burst and accompanying cytosolic Ca2+ release that occurs in the apoplast in response to the perception of biotic and abotic stresses (Kawano and Muto, 2000). The apoplastic oxidative burst and resultant ROS accumula- tion in the extracellular space is characteristic of plant cells exposed to abiotic stresses including physical and chemical shocks, insects and herbivores, symbiotic microorganisms and pathogens. Class III peroxidases may participate in SA-induced ROS metabolism. It has been suggested that SA could act as an e– donor for Prx, a pro- cess that would generate SA radicals (Kawano et al., 2004). Some of the SA-dependent ROS production in plant cells might therefore depend on the interaction between SA and Prx as well as the activa- tion of NADPH oxidases (Almagro et al., 2009). SA is also considered to be an inhibitor of the respiratory alternative oxidase (Hayat et al., 2007). The activation of NADPH oxidases also serves to suppress the spread of pathogen- and SA-induced cell death (Pogány et al., 2009). The plant plasma membrane NADPH oxidases were discovered on the basis of their sequence similarity to the mammalian respira- tory burst NADPH oxidase subunit gp91phox and are therefore also called respiratory burst oxidase (RBOH) proteins. In Arabidopsis the 10 genes encoding these proteins are called rboh with different genes designated by letters from A to J. The RBOH proteins fulfil different functions in the regulation of plant responses to environ- mental stresses. For example, RBOHD triggers death in leaf cells that are under fungal attack but it simultaneously inhibits death in the neighbouring cells by the suppression of SA and ET (Pogány et al., 2009). Mutants defective in the RBOHD or RBOHF proteins show enhanced SA-induced cell death (Torres et al., 2005). The Arabidopsis lesion simulating disease 1 (lsd1) mutant is characterised by a ROS-dependent spreading cell death phenotype when grown under high or continuous light or upon infection with avirulent pathogens (Aviv et al., 2002; Dietrich et al., 1997; Jabs et al., 1996; Kliebenstein et al., 1999; Mateo et al., 2004, 2006). Mutations in SA signalling genes such as Phytoalexin Deficient 4 (PAD4) and Enhanced Disease Susceptibility 1 (EDS1) block runaway d Exp c M s a c t e r s p i T b o e S p i S N T m i c e o t l f g m P t 2 a 2 g s t b e c a 2 f i r T t ( r 2 m w o i v K e B s p C.G. Bartoli et al. / Environmental an ell death in lsd1 (Aviv et al., 2002; Rustérucci et al., 2001). oreover, crossing lsd1 with a transgenic line carrying a bacterial alicylate hydroxylase (NahG) or in npr1 mutants attenuated run- way cell death after SA treatment. It is a redox-sensitive cell death ontroller in plants exposed to stresses such as, high light and low emperatures (Epple et al., 2003; Huang et al., 2010; Mühlenbock t al., 2008). The amino acid sequence of the encoded LSD1 protein eveals that it contains two highly conserved Cys–Gly–His–Cys ites within the zinc fingers that are characteristic of protein disul- hide isomerases, which regulate the formation, reduction and somerisation of disulphide bonds associated with protein folding. he presence of this sequence suggests that the LSD1 protein may e regulated by Trx, Grx and glutathione, like another component f the SA signalling pathway, NPR1 (Wildermuth et al., 2001; Mou t al., 2003; Tada et al., 2008). NPR1 is an essential component of A signalling cascade, which induces SAR. In the cytosol, NPR1 is resent in an inactive oligomeric complex that is formed through ntermolecular disulphide bonds. The SA-dependent induction of AR requires monomerisation of the oligomeric cytosolic protein PR1 (Després et al., 2003; Mou et al., 2003; Laloi et al., 2004a,b). he monomerisation process reveals a nuclear localisation signal otif that allows the protein to localise to the nucleus, where it nteracts with TGA transcription factors that have a minimum as-1 is-element of TGACG, and are themselves redox-sensitive (Mou t al., 2003). There are 10 TGA transcription factors in Arabidopsis f which seven (TGA1–TGA7) have been characterised with respect o their interaction with NPR1 (Kesarwani et al., 2007). Glutathione (Mou et al., 2003) and Trxh (Tada et al., 2008) are inked in the activation of this pathway. Exogenous GSH can mimic ungal elicitors in activating the expression of defence-related enes such as, PR1 (Gómez et al., 2004). Moreover, glutathione accu- ulation is triggered by pathogen infection (Vanacker et al., 2000; arisy et al., 2006) in a similar manner to that reported following he application of SA or biologically active SA analogues (Mou et al., 003). SA-deficient NahG Arabidopsis lines showed increased GSSG ccumulation and enhanced salt stress tolerance (Borsani et al., 001). In contrast, SA-deficient NahG rice lines had a decreased lutathione pool and showed increased susceptibility to high light tress (Kusumi et al., 2006). SA-inducible genes include certain glu- athione S-transferases (GST), some of which are also considered to e markers for increased H2O2 (Vanderauwera et al., 2005; Queval t al., 2007, Chaouch et al., 2010). In the absence of pathogen challenge, NPR1 is continuously leared from the nucleus by the proteasome, which restricts its co- ctivator activity to prevent untimely activation of SAR (Spoel et al., 009). The turnover of NPR1 is promoted by phosphorylation which acilitates its recruitment to a Cullin3-based ubiquitin ligase, which s part of the SFCcoi ubiquitin-ligase complex that is considered to egulate cross-talk between the SA- and JA-signalling pathways. he phosphorylated form of NPR1 is required for the expression of arget genes and the establishment of SAR (Spoel et al., 2009). Ascorbate-deficient Arabidopsis mutants such as vitamin C vtc)1 and vtc2 have a slow growth phenotype and enhanced basal esistance to biotrophic pathogens (Pastori et al., 2003; Pavet et al., 005; Mukherjee et al., 2010). The vtc1-1, vtc2-1, vtc3-1, and vtc4-1 utants were all more resistant to Pseudomonas syringae than the ild type plants (Mukherjee et al., 2010). Consistent with these bservations, a large number of transcripts that encode the SA- nducible proteins are constitutively expressed in the vtc1 and tc2 mutants (Kiddle et al., 2003; Pavet et al., 2005; Brosche and angasjarvi, 2012). The abundance of leaf ascorbate has a key influ- nce over both SA and JA signalling pathways (Kerchev et al., 2011; rosche and Kangasjarvi, 2012). Glutathione and glutathione-mediated redox control of SA ignalling are considered to be important in the regulation of rocesses that underpin acclimation to high light as well plant erimental Botany 94 (2013) 73– 88 77 immune responses (Dong, 2004; Pieterse and Van Loon, 2004; Chang et al., 2009). This pathway has also been studied intensively in mutants that are deficient in the catalase 2 (cat2) isoform, which is important in the removal of H2O2 generated by photorespiration (Chaouch et al., 2010; Queval et al., 2011). The cat2 mutants show a conditional cell death phenotype with induction of associated defence responses that is completely dependent on SA (Chaouch et al., 2010). Moreover, SA-dependent cell death was abolished by myo-inositol, which also eliminated the H2O2-dependent repro- gramming of pathogen defence responses (Chaouch and Noctor, 2010). The leaves of the cat2 mutants showed a large increase in the total glutathione (GSH plus glutathione disulphide {GSSG}) pool with a markedly increased GSSG content under photorespiratory conditions (Chaouch et al., 2010; Queval et al., 2007). GSSG accu- mulation was increased even further in comparison to the wild type plants in cat2 gr1 double mutants that are deficient in both the major catalase and also the cytosolic glutathione reductase, GR1 (Mhamdi et al., 2010). Taken together, these observations have led to the concept that glutathione is a modulator of SA and JA signalling pathways (Noctor et al., 2012). More information was provided on the interaction between SA and oxidant signalling pathways by the analysis of mutants that are defective in singlet oxygen signalling (Wagner et al., 2004). Singlet oxygen is mainly produced in plants by the photosys- tem II reaction in the chloroplasts (Triantaphylides and Havaux, 2009) where it is quenched by �-tocopherol (Shao et al., 2008). Like H2O2, singlet oxygen is a highly reactive form of oxy- gen, whose accumulation triggers programmed cell death (Danon et al., 2005; Triantaphylides and Havaux, 2009). However, sin- glet oxygen-induced programmed cell death is abolished in the Arabidopsis fluorescent (flu) mutant that accumulates the sensitiz- ing compound, protochlorophyllide, an intermediate in chlorophyll biosynthesis in the dark (op den Camp et al., 2003). When trans- ferred into the light after a period of darkness, the flu mutant produces large amounts of 1O2 leading to major changes in nuclear gene expression (op den Camp et al., 2003). This path- way requires the expression of the EXECUTOR1 and 2 genes, which encode thylakoid proteins (Lee et al., 2007). The inactivation of the EXECUTER1 chloroplast protein was sufficient to prevent sin- glet oxygen-induced cell death in flu seedlings and prevent singlet oxygen-induced growth inhibition in mature plants (Wagner et al., 2004). Singlet oxygen selectively activates nuclear genes that are either not responsive or are less responsive to superoxide or H2O2 suggesting that activation by 1O2 might require promoter ele- ments that are different from those used by H2O2. When the flu mutant seedlings were made SA-deficient by expression of salicy- late hydroxylase (NahG), they were partially protected from the singlet oxygen-mediated cell death indicating that SA is required for the induction of the cell death programme. 4. Ethylene The metabolic precursor of ethylene (ET; C2H4), 1- aminocyclopropane-1-carboxylic acid (ACC), is produced by the S-adenosylmethionine pathway. The first committed step of ET biosynthesis is the conversion of S-adenosylmethionine to ACC by S-adenosyl-l-methionine methylthioadenosine-lyase (ACC synthase). ET participates in many aspects of plant biology from germination to dormancy, ripening and senescence, and the regulation of stomatal closure, as well as defences against biotic and abiotic stresses (Bleecker and Kende, 2000; Lin et al., 2009). Like ABA, ET signalling pathways are crucial to the survival of adverse environmental conditions and it is involved in the control of growth (Achard et al., 2003) as well as stress tolerance. For example, drought induced accumulation of the ET precursor, 7 d Exp 1 n E A r a a fi d o t r c b d d i s s H g h U m E a k I s ( C t E t f i p i s e b s t r t r c u r B f b D v b a P s r i G e 8 C.G. Bartoli et al. / Environmental an -aminocyclopropane-1-carboxylate and the activation of ET sig- alling lead to a reversible arrest of cell cycle (Skirycz et al., 2011). T accumulation leads to an inhibition of cyclin-dependent kinase activity in a manner that was independent of EIN3 transcriptional egulation (Skirycz et al., 2011). However, ET production can have negative effect on crop production because it triggers senescence nd hastens maturity, shortening the grain filling period and lling rate. ET also increases the incidence of embryo abortion, ecreasing parameters such as thousand-grain weight. ET induces ROS generation and H2O2 stimulates the expression f ET-responsive proteins and of the enzymes involved in ET biosyn- hesis (Vandenabeele et al., 2003). Moreover, ROS-dependent plant esponses often require ET sensitivity. For example, in the pro- ess of leaf abscission, a special layer of cells is formed that must e destroyed to allow removal of the leaf. The programmed cell eath pathways occurring in this layer depend on NADPH oxidase- ependent H2O2 generation triggered by ethylene. Antioxidants or nhibitors of ET or NADPH oxidases block the progress of leaf abscis- ion (Sakamoto et al., 2008). ET acts upstream in ROS-dependent ignalling cell death responses (Chae and Lee, 2001). ET enhances 2O2 production in plant cells that are destined to undergo pro- rammed cell death (De Jong et al., 2001; Moeder et al., 2002). ET as been implicated in the sensitivity of plants to stresses such as V-B exposure (Mackerness et al., 1999) and exposure to heavy etals such as lithium (Bueso et al., 2007). Mutants with reduced T sensitivity are less sensitive to lithium, which triggers H2O2 ccumulation (Bueso et al., 2007). ET perception involves a family of two-component histidine inase-like receptors that can form both homo- and heterodimers. n the absence of ET, these receptors repress ET responses by ignalling through the negative regulator Raf-like MAPKK, CTR1 Kieber et al., 1993). Once ET is perceived the kinase domain of TR1 is inactivated and this allows signalling to proceed through he EIN2 protein and the DNA binding proteins EIN3 and EIL1. IN3 activates ethylene response factor 1 (ERF1) transcription fac- or, which is a GCC-box-binding transcription activator responsible or the activation of the transcription of ET-responsive genes. EIN3 s regulated by the stability and turnover of the ERF1 signalling athway, which is regulated by a MAP kinase signalling cascade nvolving MAPK3 and MAPK6. The Arabidopsis MAPK3 and MAPK6 ignalling pathways are also involved in H2O2 signalling (Kovtun t al., 2000). The ERF proteins, which are defined by a conserved DNA- inding domain, belong to the AP2/ERF transcription factor uperfamily. They contribute to the regulation of gene transcrip- ion regulation of plant growth and development, particularly in elation to environmental stress. Overexpression of ERF1 activates he expression of genes related to pathogenesis and enhances the esistance to pathogens such as Botrytis cinerea and Plectosphaerella ucumerina. ERF proteins have also been implicated in the reg- lation of plant metabolism, for example they are considered to egulate the synthesis of JA, gibberellins, ET, lipids and waxes (De oer et al., 2011). Stomatal closure required the integrated cooperation of dif- erent signalling pathways including those mediated by ABA, ET, rassinosteroids, H2O2 and NO (Desikan et al., 2005; Wilkinson and avies, 2010). H2O2 synthesis in the guard cells involves the acti- ation of NADPH oxidase isoforms AtrbohD and AtrbohF that can e triggered by either ABA or ET, with downstream functions for range of signalling components such as ABA insensitive 2 (ABI2) P and MPK3. The NADPH oxidase-deficient AtrbohD or F mutants how decreased ET–induced stomatal closure and similarly, the ET eceptor mutants etr1-1 and etr1-3 that are ET-insensitive have mpaired H2O2-dependent stomata closure (Desikan et al., 2006; refen et al., 2008). While ABA can induce H2O2 accumulation in tr1-1, the H2O2 that is produced does not result in stomatal closure erimental Botany 94 (2013) 73– 88 in the etr1-1 mutant, which is hence insensitive to H2O2 (Desikan et al., 2006). Histidine kinases are considered to be H2O2 sensors and H2O2 might interact with the ETR1 histidine kinase receptor (Desikan et al., 2006). The stomata of the ET signalling mutants, ein2-1 and arr2, do not close in response to either ET or H2O2 but do generate H2O2 following ET stimulation. The ET signalling path- way may therefore involve components that are upstream of EIN2 and ARR2 that activate AtrbohD and F activity. Moreover, ET sig- nalling that is downstream of EIN2 and ARR2 is also required for H2O2 perception. NO-associated H2O2 synthesis also participates in the interaction between the ABA and ET signalling pathways that regulate stomatal closure. ABA induces NO synthesis in guard cells but this requires prior activation of AtrbohD and F and NADPH oxidase-dependent H2O2 synthesis (Bright et al., 2006). Transgenic plants constitutively expressing dehydroascorbate reductase have a higher ratio of reduced to oxidised ascorbate and they show decreased stomatal closure, consistent with the key role of ROS in stomata opening (Chen and Gallie, 2004). Rel- atively little is known about the regulation of ascorbate synthesis genes but the F-box protein AMR1 was shown to negatively reg- ulate the expression of genes encoding pathway enzymes (Zhang et al., 2009). Similarly, members of the AP2/ERF transcription fac- tor family such as Sub1A and JERF3, which enhance stress tolerance by activation of antioxidant enzyme-related pathways (Wu et al., 2008; Fukao et al., 2011), may also regulate the expression of genes encoding ascorbate synthetic enzymes. The ein2-1, ein3-1 and ein4 mutants have higher leaf ascorbate than the wild type plants, while ctr1-1 mutants that are defective in the ET-regulated constitutive triple response factor have lower leaf ascorbate levels (Gergoff et al., 2010a). Tomato fruits with defects in ET sensitiv- ity also display greater ascorbate accumulation (Alba et al., 2005). Treatment with ethephon produces a rapid decrease in leaf ascor- bic acid contents. Moreover, post-harvest treatment of fruits and vegetables with ET inhibitors delays senescence and extends shelf life, consistent with a higher accumulation of ascorbic acid or other antioxidants (Watkins, 2006; Gergoff et al., 2010b). Fruit and veg- etables that have a climacteric ripening process show a high ET peak and enhanced ET sensitivity during organ ripening that is followed by decreased antioxidant contents (Bartoli et al., 1996; Egea et al., 2010). 5. Jasmonic acid JA belongs to a group of compounds called oxylipins that are formed via oxygenation of fatty acids. JA synthesis begins in the chloroplasts where lipoxygenases convert linolenic acid into hydroperoxylinolenic acid. A range of different metabolites are produced from hydroperoxylinolenic acid including cis + -12- oxophytodienoic acid (OPDA). OPDA can either be retained in the chloroplasts where it is used as a precursor for the synthesis of other oxylipins or it can be transported to the peroxisomes. Similar auxin, the last step of JA synthesis occurs within the peroxisomes, where it is dependent on b-oxidation. JA and related compounds regulate plant responses to wound- ing and necrotrophic pathogens (Devoto and Turner, 2005). JA-mediated reprogramming of gene expression is perhaps been best characterised in relation to plant–pathogen interactions (Overmeyer et al., 2003; Pauwels et al., 2009). JA can act synergistically with ET in the regulation of defences against necrotrophic pathogens and herbivorous insects. Chewing insects and necrotrophic pathogens activate JA and ET-dependent defence pathways (Kerchev et al., 2012). In contrast, SA-dependent defence pathways are more often activated in response to biotrophic pathogens. Antagonistic and synergistic relationships between the SA and JA/ET defence pathways have been reported. For example, JA d Exp c s u c d t d i J p a e e 2 d p a t i l s i g a t h J g 2 l t w l o a o t p t w t a P i g T p a 2 l w b 2 t D a i i P a p d e C.G. Bartoli et al. / Environmental an an antagonise the spread of programmed cell death through the uppression of SA biosynthesis and signalling, and also by atten- ation of ET sensitivity (Overmeyer et al., 2003). There is also onsiderable evidence for cross talk between the hormone-induced efence-signalling pathways and the redox signalling pathways hat might be important in defining the plant defence strategy epending on the type of attacker (Kerchev et al., 2012). The ncreased ozone sensitivity of JA mutants supports the view that A participates in the containment of the ROS-dependent lesion ropagation. Similarly, JA may influence ET-dependent lesion prop- gation by reducing the ET-dependent ROS generation (Overmeyer t al., 2003). The JA signalling pathways control growth through ffects on DELLA proteins (see Section 8; Robert-Seilaniantz et al., 011). DELLA proteins physically interact with JASMONATE ZIM- omain (JAZ) proteins that activate JA signalling pathways by reventing AtMYC2 repression (Robert-Seilaniantz et al., 2011). The JAZ proteins repress JA signalling pathways when JA levels re low, through interactions with the MYC2 transcription fac- or (Thines et al., 2007). The isoleucine conjugate of JA promotes nteraction between JAZ proteins and the SCFcoi1 ubiquitin ligase, eading to JAZ degradation via the 26S proteasome. JA synthesis timulates JAZ binding to COI1, which is an F-box protein that nteracts with JAZ transcriptional repressors. Binding to COI1 trig- ers JAZ degradation via the ubiquitin/26S proteasome pathway, nd releasing MYC2 from repression (Chung and Howe, 2009). In his way, JAZ repressors are removed and transcription factors that ad previously been bound to JAZ proteins are able to stimulate A-dependent gene expression. The expression of JA-responsive enes is responsive to the abundance of ascorbate (Kerchev et al., 011) and to the redox state of the glutathione pool. For example, a arge number of JA-responsive genes are repressed in gr1 mutants hat lack the cytosolic/peroxisomal form of glutathione reductase, hereas gr1 cat2 double mutants that lack both GR and the major eaf form of catalase show H2O2-induced expression of these and ther JA-associated genes (Mhamdi et al., 2010). Increases in tissue JA contents and the expression of JA- ssociated defensive proteins occurs in response to a wide range f environmental stimuli including pathogen attack, touch, elicita- ion, wounding and osmotic stress (Rao et al., 2000). JA signalling athways can be systemic in nature. For example, JA accumula- ion and the expression of JA-responsive genes that occur after ounding to a single leaf is also observed in leaves that are dis- ant from the wound site (Koo et al., 2009). Heavy metal stress lso triggered JA accumulation in leaves of Arabidopsis thaliana and haseolus coccineus (Maksymiec et al., 2005). JA has been implicated n NADPH oxidase activation, with H2O2 acting as a second messen- er regulating the defence response (Orozco-Cárdenas et al., 2001). he JA-response genes include antioxidants and associated defence roteins such as genes encoding enzymes involved in ascorbate nd glutathione synthesis (Xiang and Oliver, 1998; Wolucka et al., 005). Wounding like JA favours increased ascorbic acid accumu- ation but this effect varies between plant species. For example ounding and JA lead to higher ascorbic acid in Arabidopsis leaves, ut they lead to decreased ascorbic acid levels in tomato (Suza et al., 010). However, water stress-induced JA accumulation in A. crista- um increased the transcripts and activities of APX, GR, MDHAR, HAR, GalLDH, as well as enhancing the contents of ascorbic acid nd glutathione (Shan and Liang, 2010). Ascorbic acid deficiency n the Arabidopsis vtc1-1, vtc2-1, and vtc3-1, mutants leads to ncreased levels of ABA (Pastori et al., 2003) and SA, involving the AD4, EDS5 and NPR1 signalling pathways (Mukherjee et al., 2010) s well as ABA-dependent repression of JA-dependent signalling athways (Kerchev et al., 2011). The JA-dependent stimulation of antioxidant and associated efence proteins explain why wounding or applying JA before the xposure to the atmospheric oxidant ozone decreases the extent erimental Botany 94 (2013) 73– 88 79 of ozone-induced programmed cell death in tobacco (Örvar et al., 1997) and the hybrid poplar (Koch et al., 2000). Methyl-JA (a bio- logically active derivative of JA) also decreased ozone-induced cell death in Arabidopsis (Rao et al., 2000). In this case, the exogenous application of methyl-JA attenuated ozone-induced H2O2 accumu- lation (Rao et al., 2000). However exposure to ozone resulted in the development of large lesions in the JA-insensitive mutant, jar1, and in the fad3/7/8 mutant that is defective in JA biosynthesis (Rao et al., 2000). Such results demonstrate that JA-dependent signalling pathways play an important role in the regulation of programmed cell death that is triggered in response to ozone pollution and expo- sure to other oxidants. Ozone treatments result in a rapid induction of antioxidant genes in the Arabidopsis wild type but not in the JA-deficient (opr3) mutants (Sasaki-Sekimoto et al., 2005). 6. Strigolactones Strigolactones (SLs) are signalling molecules that are synthe- sised from carotenoids in plastids mainly in the lower parts of the stem and in the roots in response to metabolic and environmen- tal triggers (Domagalska and Leyser, 2011). They are important in the control of interactions with other organisms in the environ- ment (Dun et al., 2009) such as mycorrhizal fungi (Bouwmeester et al., 2007; Xie et al., 2010) and parasitic plants (Striga sp. and Oro- branche sp). SLs are transported by members of the ATP-binding cassette (ABC) transporter family (Kretzschmar et al., 2012) and they function downstream of auxin in the control shoot and root branching (Gomez-Roldan et al., 2008; Umehara et al., 2008). They are considered to second messengers in auxin signalling pathways that interact with auxin in a dynamic feedback loop in the control of organ development. By restricting auxin transport in a systemic and local manner they cause auxin accumulation to levels that inhibit growth for example in buds to control of axillary shoot branching. They also influence senescence and photomorphogen- esis (Umehara et al., 2008; Gomez-Roldan et al., 2008; Tsuchiya et al., 2010; Ruyter-Spira et al., 2011). It is possible that the SL sig- nalling pathway, like that of auxin and other hormones, produces ROS as second messengers, as suggested in Fig. 3. Several studies have indicated that SLs interact directly with the redox signalling network (Woo et al., 2004) but the precise nature of this interaction is not yet understood. For example, the delayed senescence mutant, ore9, is more tolerant to oxidative stress than the wild type. ORE9 is a homologue of MAX2, which is a component of the SL signalling pathway (Woo et al., 2001; Stirnberg et al., 2002). SL signalling also controls root architecture (Kapulnik et al., 2011a,b; Ruyter-Spira et al., 2011). The SL-dependent control of root branching is lost in the max2 mutants, which are insensitive to the synthetic SL, GR24 (Fig. 4A). Treatment with GR24 causes some changes in the abundance of ascorbate and glutathione in the roots and shoots of Arabidopsis seedlings (Fig. 4B) but it has little or no impact on the activities of antioxidant enzymes such as catalase or superoxide dismutase. 7. Abscisic acid The elucidation of the pathway of ABA synthesis in plants was greatly aided by the characterisation of mutants, particularly those that are impaired in zeaxanthin epoxidation, which were first iden- tified by an ABA-deficient (Schwartz et al., 2003). While a “direct pathway” of synthesis had originally been proposed in which ABA is derived from farnesyl diphosphate, it is now generally accepted that ABA is produced in plants predominantly by an “indirect path- way” involving the oxidative cleavage of a 9-cis-epoxycarotenoid (C40) to produce xanthoxin (C15) and a C25 by-product the cleavage of carotenoids (Schwartz et al., 2003). 80 C.G. Bartoli et al. / Environmental and Exp Fig. 3. A simple representation of the strigolactone synthesis pathway, showing the mutations that are available in either the synthesis pathway (max3, max4 and max1) or in signalling (max2) in A. thaliana. This scheme suggests that SLs may regulate the production or signalling of ROS. Fig. 4. (A) Schematic comparison of the effects of the strigolactone analogue, GR24, on l strigolactone signalling mutant, max2-1. (B) Effect of GR24 on the the leaf total glutathio oxidised forms + DHA) pools of 8-day-old A. thaliana seedlings. erimental Botany 94 (2013) 73– 88 ABA is a positive regulator of leaf senescence that accumu- lates in response to stresses that involve water deficits, such as drought, salt or temperature extremes leading to a reprogramming of gene expression and adaptive responses such as stomatal closure and accumulation of osmo-compatible solutes (Chandrasekar et al., 2000). ABA triggers NADPH oxidase dependent ROS production that is important in mediating the closure of stomata and the regula- tion of MAPKinase signalling cascades (Guan et al., 2000; Pei et al., 2000; Zhang et al., 2001). ABA-induced H2O2 production by the plasma membrane NADPH oxidases, RbohD and RbohF, leads to the activation of calcium-permeable channels, the increase in cytoso- lic Ca2+ causing stomata closure (Kwak et al., 2006). This activation is impaired in the ABA-insensitive gca2 mutants (Pei et al., 2000). Moreover, high ascorbic acid concentrations in guard cells made the stomata less responsive to addition of H2O2 or ABA (Chen and Gallie, 2004). ABA-induced H2O2 production by RbohD and RbohF is also important in the induction of plant defence responses (Torres and Dangl, 2005; Torres et al., 2002). ABA-mediated activation of the OST1 protein kinase is also a key component of ROS generation in ABA-signalling. Mutations in ost1 inhibit ABA-induced ROS production in guard cells (Kwak et al., 2006). H2O2 inactivates the ABI1 and ABI2 type PP2Cs that func- tion as negative regulators of the ABA signalling pathway described above (Bailly et al., 2008). PP2Cs are therefore targets for ROS sig- nalling, particularly in the co-ordinate regulation of ABA-mediated responses (Kwak et al., 2006). The PYR/PYL/proteins, which are important ABA receptors, function upstream of the type 2C protein phosphatase (PP2Cs)- SNF1-related protein kinase 2 (SnRK2) protein kinase complexes and ROS production in the regulation of ABA-mediated func- tions. SnRK2 proteins are major regulators of ABA signalling in the control of plant development and responses to water stress. The ABA-activated protein kinase (AAPK) and the related SRK2E/OST1/SnRK2.6 protein kinase regulate the activities of anion channels and stomatal closure in response to ABA upstream of ROS production. Phosphorylated SnRK2 is required for the acti- vation of ABA-induced gene expression. When ABA binds to the PYR/PYL/proteins, the resultant complex sequesters PP2Cs in a way that inhibits their phosphatase activities. The inhibition of PP2Cs allows the SnRK2 protein kinase to activate downstream targets including transcription factors such as AREB/ABF bZIP proteins and anion channels. ABA-induced ROS production and ABA activation of Ca2+ channels are impaired in Arabidopsis mutants that are ateral root development in 8-day-old A. thaliana (Colombia 0) seedlings and in the ne (reduced glutathione plus glutathione disulphide) and ascorbate (reduced plus d Exp d c s S w s w r s 2 d c t e t l i n a s 2 c J a c a ( l c m r i t i A t a c 2 a A s ( p t t p i s r v t i o t n o s l 2 b e C.G. Bartoli et al. / Environmental an efective in PP2C (Murata et al., 2001). The SnRK2-interacting cal- ium sensor, which is important in ABA-mediated regulation of eed germination, probably functions by negative regulation of nRK2 activity (Bucholc et al., 2011). The heterotrimeric protein phosphatase 2A (PP2A) complex, hich is comprised of a catalytic subunit and regulatory A and B ubunits that modulate enzyme activity and mediate interactions ith other proteins also plays a key role in the control of basal epression of defence responses through the Constitutive Expres- or of Pathogenesis-Related Genes5 (CPR5) pathway (Trotta et al., 011). The CPR5 pathway, which functions upstream of SA in NPR1- ependent disease resistance, appears to have multiple roles in ell signalling and is involved in cell wall biogenesis, disease resis- ance, cell proliferation, cell death and sugar sensing (Brininstool t al., 2008). Mutants defective in CPR5 constitutively express sys- emic acquired resistance (SAR) and forming spontaneous HR-like esions. Several PP2A mutants have been characterised to date ncluding the roots curl in naphthylphthalamicacid1 (rcn1) and ton- eau2 (ton2)/fass/gordo mutants. The rcn1 mutant shows altered uxin transport, inhibition of ET synthesis and an insensitivity of tomatal closure in response to blue light, ABA and JA (Kwak et al., 006; Tseng and Briggs, 2010). It also exhibits premature senes- ence, H2O2 accumulation and constitutive activation of SA and A-dependent defence responses (Trotta et al., 2011). Further evidence in support of the concept that there is a close ssociation between redox signalling and ABA signalling pathways omes from studies on the A. thaliana vtc1 and vtc2 mutants that ccumulate lower levels of ascorbate than the wild type plants Pastori et al., 2003; Kiddle et al., 2003; Kerchev et al., 2011). The eaves of the low ascorbate mutants have increased ABA levels ompared to the wild type plants and they show a reprogram- ing of gene expression that is characteristic of ABA signalling esponses (Pastori et al., 2003; Kiddle et al., 2003). Moreover, there s significant overlap between the transcriptome reprogramming in he vtc1 and vtc2 mutants and the abi4 mutants that are deficient n the nuclear localised Apetala 2-type (AP2) transcription factor, BSCISIC ACID (ABA)-INSENSITIVE-4 (ABI4). ABI4 is important in he ABA-dependent control of seed development and germination nd also in orchestrating plant growth responses to variations in arbon/nitrogen availability (Signora et al., 2001; Kerchev et al., 011). The negative effect of sucrose on ascorbate synthesis and ccumulation was lost in the abi4 mutant (Yabuta et al., 2007). BA synthesis and signalling are not only important factors in the low growth phenotype observed in the vtc1 and vtc2 mutants Pastori et al., 2003) but the ascorbate-dependent regulation of lant growth has an absolute requirement for the ABI4 transcrip- ion factor (Kerchev et al., 2011). In the absence of a functional ABI4 ranscription factor the low ascorbate-dependent slow growth henotype is not expressed. Thus, like ABI1 and ABI2, ABI4 fulfils mportant functions in stress signalling cascades involving ROS as econd messengers (Kerchev et al., 2011). Moreover, the decreased edox buffering capacity arising from low ascorbate availability in tc1 and vtc2 mutants drives gene expression in a similar manner o that occurring when ABI4 is not functional (Kerchev et al., 2011). ABA can have positive and negative effects on plant–pathogen nteractions depending on the nature (necrotrophic and biotrophic) f the infection (Robert-Seilaniantz et al., 2011). The activa- ion of ABA signalling pathways promotes the susceptibility to ecrotrophic pathogens. For example, ABA decreases the resistance f soybean to incompatible nonpathogenic strains of Phytophthora ojae (Ward et al., 1989). Similarly, drought-induced ABA accumu- ation in Arabidopsis decreases resistance to avirulent Pst (Mohr, 003). Conversely, ABA treatment enhanced the resistance of Ara- idopsis to biotrophic pathogens (Ton et al., 2009). ABA accumulation was associated with enhanced antioxidant nzyme activities in germinating wheat seedlings subjected to mild erimental Botany 94 (2013) 73– 88 81 osmotic stress (Agarwal et al., 2005). ABA treatment of barley aleu- rone cells increased the activities of catalase, APX and superoxide dismutase and the susceptibility to ROS-induced programmed cell death was decreased (Fath et al., 2001). Similarly, ABA treatment increased the catalase, APX and superoxide dismutase activities of maize seedling together with a beneficial effect on the contents of ascorbate, glutathione, �-tocopherol and carotenoids (Jiang and Zhang, 2002b). Similar effects were observed in leaves exposed to moderate water stress (Jiang and Zhang, 2002a). ABA can inhibit GA biosynthesis and its action is often antagonis- tic to GA, the ratio of ABA to GA being a fundamental determinant of growth or quiescence (Ross et al., 2011). For example, the GA- regulated DELLA protein called RGL2 inhibits seed germination by stimulating ABA synthesis and the activity of ABA INSENSITIVE5 (ABI5), which is a basic domain/leucine zipper transcription factor. Genetic studies have shown that RGL2 is epistatic to the ATP bind- ing cassette (ABC) transporter called COMATOSE (CTS, also called PXA1 and PED3), which is involved in the import of substrates into the peroxisomes for b-oxidation pathways. The block on seed ger- mination caused by a mutation in CTS cannot be rescued by GA, but germination is rescued by mutations at the ABI5 locus, provid- ing genetic evidence for a direct link between the pathways (Kanai et al., 2010). Redox regulation of the balance between ABA and GA-signalling pathways has also been suggested to influence ger- mination (Liu et al., 2010) and other processes such as dormancy and floral induction (Barth et al., 2004). 8. Gibberellins Gibberellins are cyclic diterpene compounds that regulate plant growth and development. They stimulate elongation or expan- sion of organs via enhancement of cell elongation and, in some cases, also cell division. Furthermore, GAs may induce develop- mental switches, such as between the juvenile and adult phases or between vegetative and reproductive development. They have also been implicated in the function of the meristem, where they are thought to promote differentiation and suppress the maintenance of stem cells. GA functions are also responsive to environmental cues, including changes in light conditions, temperature or stress, (Yamaguchi, 2008) allowing GAs to translate these extrinsic sig- nals into developmental changes. GA synthesis is also influenced by ascorbate availability. Ascorbate is a co-factor in the catalysis of 2-oxoacid-dependant dioxygenase (2ODD) reactions and the activ- ities of these enzymes are enhanced by the addition of ascorbate in vitro (Arrigoni and De Tullio, 2000). Dioxygenases are impor- tant in the final stages of GA synthesis, where GA12 is converted to bioactive GAs (Hedden and Kamiya, 1997). GAs mediate growth in response to environmental signals by relieving the constraints on gene expression imposed by a fam- ily of growth-repressing regulators, the nuclear growth-repressing DELLA proteins, as mentioned above (Peng et al., 1999; Harberd et al., 2009). GAs and auxin have many overlapping functions in the control of organ expansion. While auxin also appears to control DELLA stability independent of its regulation of GA, the two sig- nalling pathways interact, with auxin, controlling growth at least in part by modulation of the GA signalling cascade. GA levels was first linked to stress protection in an analysis of the responses of wild type and dwarf barley near-isogenic lines to heat stress (Vettakkorumakankav et al., 1999). Arabidopsis mutants that have a reduced GA content or containing a GA-insensitive form of DELLA proteins are more salt tolerant than wild-type plants, while plants with loss-of-function DELLA mutations have increased sus- ceptibility to salt stress (Achard et al., 2006). It is now generally accepted that the DELLA proteins, which are transcriptional reg- ulators that restrain plant growth, play a key role in the control 8 d Exp o G p m ( t a a c a e t t e c f e l a d o d a f t o i d p y v m h B a n a e p S r p T o m D m ( t r t i o a e b R t r T D i d 2 C.G. Bartoli et al. / Environmental an f growth and tolerance in response to biotic and abiotic stresses. A promotes growth by stimulating the destruction of the DELLA roteins, which accumulate in plants exposed to adverse environ- ental conditions to restrain growth and enhance plant survival Achard et al., 2003, 2006; Harberd et al., 2009). Plants exposed o salt stress had increased DELLA contents and showed less ROS ccumulation and enhanced expression of genes encoding for ntioxidant enzymes (Achard et al., 2008). The DELLA proteins also ontribute to pathogen resistance because the GA/DELLA pathways lter the balance between the SA and JA/ethylene signalling (Achard t al., 2008). Higher DELLA protein contents favour increased resis- ance to necrotrophic pathogens (Achard et al., 2008). Moreover, he GA-deficient ga1-3 Arabidopsis mutants that have higher lev- ls of DELLA proteins are much less susceptible to ROS-dependent ell death (Achard et al., 2008). The accumulation of DELLA proteins can remove the potential or cell proliferation (Skirycz et al., 2011). Stress conditions that nhance ABA/GA ratios favouring DELLA protein accumulation and ower ROS levels (Finkelstein et al., 2008). High ABA/GA ratios can lso induce dormancy in tubers, buds and seeds. For example, ABA ecreased ROS production in rice seeds leading to a repression f ascorbate and GA accumulation (Ye et al., 2012). In contrast, ormancy release is stimulated by GA and ROS but inhibited by ntioxidants (Oracz et al., 2009). The ‘Green Revolution’ that improved worldwide wheat yields rom the 1960s was based upon a combination of new varieties and he increased use of fertilisers and pesticides. An important feature f the new varieties was reduced height: the semi-dwarf plants had ncreased yield, as less of the plants’ energy was wasted on pro- ucing straw and more went into the harvested grain. The shorter lants were also stronger and more capable of bearing the increased ields without lodging (falling over) in wind and rain. These dwarf arieties of wheat carried genes (called “Reduced Height” or Rht) that ade them unresponsive to GA, which normally increases stem eight. The most commonly used alleles of these genes are Rht- 1b and Rht-D1b (previously called Rht1 and Rht2) semi-dwarfing lleles derived from Norin 10, which reduce sensitivity to endoge- ous GAs. Wheat plants carrying altered function Rht-B1b/Rht-D1b lleles of Rht-1 genes (dwarf plants) show increased antioxidant nzymes and retain higher chlorophyll contents when subjected to otassium deficiency (Moriconi et al., 2012). Mutations in the DELLA proteins or GA (GA3) treatment increase A biosynthesis and signalling (Robert-Seilaniantz et al., 2011). A ole for GA in SA-dependent responses has also been reported in lants subjected to abiotic stress (Alonso-Ramírez et al., 2009). he application of GA3 reversed the inhibitory effect of salt, xidative, and heat stresses on the germination and establish- ent of Arabidopsis seedlings (Alonso-Ramírez et al., 2009). The ELLAs proteins also regulate JA signalling pathways. GA pro- otes JA biosynthesis in stamens by a DELLA-dependent process Robert-Seilaniantz et al., 2011). The over-expression of DREB/AP ranscription factors, which are involved in many stress responses, esults in GA-reversible growth inhibition and a reduction in bioac- ive GA concentration. Phosphate limitation-dependent changes n root architecture result, at least in part, from decreased levels f bioactive GAs. GA metabolism is influenced also by nitrogen vailability, particularly when primary nitrogen assimilation is nhanced as a result of alteration in cellular redox state caused y the absence of mitochondrial Complex I (Pellny et al., 2008). espiratory Complex I is the first enzyme of the respiratory elec- ron transport chain. It is a rotenone-insensitive NADH ubiquinone eductase that couples electron transport to proton translocation. he near homoplasmic Nicotiana sylvestris CMSII mitochondrial NA mutant is devoid of the NAD7 gene and Complex I assembly is mpaired and thus the CMSII mutants lack the major NADH dehy- rogenase of the respiratory electron transport chain (Dutilleul erimental Botany 94 (2013) 73– 88 et al., 2005). However, the mutant is able to sustain electron flow through the engagement of non-phosphorylating alternative dehy- drogenases that have a low affinity for NADH and this causes an alteration in cellular pyridine nucleotide homeostasis. The CMSII mutants show a large increase in tissue pyridine nucleotides and a much higher level of NADH available to drive NADH-requiring path- ways such as primary nitrogen assimilation (Dutilleul et al., 2005). The enhanced NAD(P)H availability also exerts a significant influ- ence over GA synthesis and signalling (Pellny et al., 2008). Nitrogen availability also influences the expression of genes encoding GA- biosynthetic enzymes, particularly the abundance of transcript for the GA-inactivating gene GA2ox and the biosynthetic gene GA3ox (Pellny et al., 2008). The CMSII mutants show slow growth pheno- type but the wild type growth phenotype can be partially restored by GA treatment (Pellny et al., 2008). The CMSII mutants also showed large changes in the levels of the GA-biosynthetic inter- mediates suggesting redox control of GA synthesis and metabolism (Pellny et al., 2008). Further evidence in support of the concept that GA synthesis and metabolism are responsive to redox con- trols comes from an analysis of the cysteine-rich GASA4 protein, which promotes GA responses (Rubinovich and Weiss, 2010). The GAST1-like proteins are considered to be involved in redox reac- tions by virtue of their cysteine-rich domain and they may regulate the redox status of specific components to promote or suppress GA- related responses. The over-expression of GASA4 suppressed ROS accumulation enhanced resistance to NO (Rubinovich and Weiss, 2010). 9. Conclusions and perspectives Accumulating evidence supports the concept that cellular redox signalling and hormone signalling pathways form an integrated redox-hormone network that regulates plant growth and defence pathways (Fig. 5). The efficient operation of this network requires extensive metabolic crosstalk and multiple points of reciprocal con- trol. For example, stomatal closure is controlled by a number of hormones including ABA, brassinosteroids and ET and it requires redox regulation through NO and ROS production (Foyer et al., 2008) but the abundance of ascorbate in the guard cells modulates hormone action (Chen and Gallie, 2004). Root architecture is also tightly controlled by the integrated action of redox and hormone- related signals (Foreman et al., 2003; Jones et al., 2007; Takeda et al., 2008; Guo et al., 2009). Drought-induced ET accumulation can quickly and reversibly cause cell cycle arrest in a manner that allows the cells to remain in a quiescent state from which they can recover when the envi- ronmental conditions improve (Skirycz et al., 2011). In this way ET accumulation in plants suffering environmental stress can reduce root and shoot growth and biomass accumulation by direct effects on growth process. ET also disrupts the ABA-mediated control of photosynthesis and leaf growth. ABA is a significant hormone in the control of drought stress because it favours stomatal clo- sure and it can limit leaf growth in order to reduce plant water loss via transpiration. ET is therefore linked to stress sensitivity in terms of limiting crop yields, by increasing leaf injury, accelerat- ing senescence. The sensitivity of stress responses may therefore be governed by the ratio between ET (together with its precursor ACC, 1-aminocyclopropane) and ABA rather than the concentra- tion of either hormone alone. A much better understanding of such interactions is essential for the improvement of crop plants and to obtain high yields under stressful environmental conditions. Programmed cell death is an important plant defence response that prevents the proliferation of pathogens. SA, JA and ET fulfil essential roles in mediating pathogen responses and they inter- act in the control of ROS generation and antioxidant enzyme C.G. Bartoli et al. / Environmental and Exp Fig. 5. Diagrammatic representation of the interactions between hormone and r o a ( t c b p 2 a d a t J l w m t p s g t s a o l c a a p t w v ( Plant Physiology 134, 178–192. edox signalling pathways in the control of growth and defence responses. Reactive xygen species (ROS). ctivities to control stress responses and cell suicide pathways Rao et al., 1997; Horváth et al., 2007; Ashraf et al., 2010). The hreshold for such signalling events is strongly influenced by ellular redox buffering capacity, as illustrated in the low ascor- ate mutants, which show enhanced resistance to biotrophic athogens (Barth et al., 2004; Pavet et al., 2005; Mukherjee et al., 010). Conversely, plants over-expressing ascorbate oxidase have more highly oxidised ascorbate redox state in the apoplast show ecreased pathogen resistance (Pignocchi et al., 2006). Glutathione lso plays a key role in the SA, JA and ET interaction, as illus- rated in the similarities between glutathione-associated genes and A-dependent gene expression (Mhamdi et al., 2010). Genes that ink glutathione and JA include those encoding GSTs and GRXs hich could link glutathione to JA signalling pathways, in a similar anner to the regulation of auxin-signalling pathways by glu- athione, as discussed above. The redox state of the glutathione ool is modulated by biotic challenges, while JA accumulation can timulate the expression of the genes encoding the enzymes of lutathione synthesis. Such findings underline the potential impor- ance of ascorbate and glutathione as regulators of redox-triggered ignalling through the SA, JA and auxin signalling pathways. The regulation of growth and defence in response to hormones nd redox signals is highly dependent on cell identity and devel- pment stage. For example, the accelerated senescence of older eaves is an important trait for plant survival under unfavourable onditions. Stress-induced ET production and ROS accumulation re observed in mature leaves exposed to stress but these changes re not found in the young leaves, which are less likely to exhibit remature senescence (Pazmiño et al., 2011). Such considera- ions have to be taken into account in post harvest physiology, here the quality and nutritional characteristics of fresh fruits and egetables have to be preserved between harvest and consumption Kader, 2002). Detachment from the plant and storage (especially erimental Botany 94 (2013) 73– 88 83 in dark and refrigerated storage) is stressful for edible organs and leads to loss of “freshness”, characteristics such as appearance and firmness (Hodges and Toivonen, 2008). Loss of freshness is impor- tant because consumers select products on this basis (Bruhn, 2002). Consumers are also interested in foods with a high nutritional qual- ity particularly vitamin, mineral and fibre contents. The acquisition of cross-tolerance characteristics by post- harvest treatments with mild stresses such as low ozone, H2O2, heat shock and UV-C, has been successfully used to extend shelf life and improve post-harvest storage. For example, heat shock has been successfully used for extending the post-harvest life of spinach leaves (Gómez et al., 2008). Heat shock signalling is depen- dent on H2O2 for increasing abiotic stress tolerance (Gechev et al., 2002). Heat shock can be used to increase the antioxidant con- tents of plant organs during storage (Zhang et al., 2005; Vicente et al., 2006; Costa et al., 2005). A better understanding of plant ROS–antioxidant–hormone interactions will facilitate the further development of these “clean technology” approaches to plant pro- duce conservation. 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