Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Senescence (at the whole plant level) or programmed cell death (PCD, at the cellular level) is one among many manifestations of ageing and death in plants, associated with (a) resource recycling; (b) resource reallocation to reproductive growth; and/or (c) adaptation to biotic/abiotic stresses. One of the oldest and most popular theories concerning ageing (often discussed in the context of animals) is the free radical theory. This theory postulates that ageing is due to the cumulative metabolic damage suffered by a living system over its entire life span through the action of reactive oxygen species (ROS) (Kirkwood & Kowald 2012). Despite significant progress in our understanding of ROS inflicted ageing, there are many unknowns, and senescence should not be attributed to a single mechanism. The diversity in mechanisms of ageing, longevity (and ultimately death) in plants becomes apparent when we study ageing in a cell-type specific context. For example, telomere stability and degradation are fundamental to replicative senescence in undifferentiated plant meristematic cells (González-García et al. 2015). Enucleated phloem sieve elements continue to live as long as their life support systems (companion cells) perform as metabolic outsourcing agents (Furuta et al. 2014). Xylem ray parenchyma cells live for >50 years in conifers, and their secret to extraordinary life spans is yet to be discovered (Nakaba et al. 2012). Yet, in all cases, ROS induced organelle damage, and depletion of plastid resources could be a necessary but not sufficient condition for all plant cells to degenerate beyond recovery and to enter senescence (Fig. 1). ROS induced metabolic impairment of plastids, and plant antioxidant strategies are particularly significant in determining the viability and longevity of photosynthetic tissues (Zentgraf & Hemleben 2007; van Doorn & Yoshimoto 2010; Khanna-Chopra et al. 2013). Plastids and mitochondria are the two locations within plant cells where electron transport, oxygen exchange and ROS formation take place (e.g. Møller et al. 2007). Chloroplast ROS are key factors that influence leaf senescence (Zapata et al. 2005). Among many different means of combating ROS in chloroplasts is the capacity of plants to synthesize a plethora of antioxidant molecules that quench ROS and prevent oxidative damage. Although mechanisms of ROS quenching by antioxidants are yet not fully clear, many experiments support the view that volatile hydrocarbons, phytohormones, enzymes and other small antioxidant molecules are involved in mitigating oxidative stress (Velikova et al. 2005; Zavaleta-Mancera et al. 2007; Weydert & Cullen 2010), and perhaps these antioxidants are orchestrated to protect leaves throughout the day (Tattini et al. 2015). The ascorbate–glutathione cycle, a major plant antioxidant defense, is activated under drought, biotic and oxidative stresses in healthy leaves and is hyper-activated in senescing leaves (Foyer & Noctor 2005; Khanna-Chopra et al. 2013). Ascorbate metabolism occurs in the cytoplasm and mitochondria, while glutathione enters chloroplasts via membrane transporters (Foyer & Noctor 2005). However, as most of the antioxidant mechanisms are, the ascorbate–glutathione cycle is active against hydrogen peroxide (H2O2). H2O2 has a relatively long life span compared with other ROS and is not actively involved in causing DNA damage (Møller et al. 2007). Singlet oxygen (1O2) and superoxide radical (O2−) are the two main ROS produced within chloroplasts that have very short half-lives (Fig. 1; Ivanov & Khorobrykh 2003). 1O2 could induce transcriptional activation of glutathione production (Leisinger et al. 2001). However, both 1O2 and hydroxyl radical (HO−) need quick intervention. Thus, antioxidants produced de novo (i.e. regulated by changes in photosynthesis) are important in preventing 1O2 from causing significant harm to the photosystems, chloroplast genome and proteins, eventually delaying cell death (Danon et al. 2005; Hideg et al. 2007). One of the pathways that contributes immensely to the de novo antioxidant pool of the plastids is the methylerythritol phosphate (MEP) pathway (Fig. 2; Peñuelas & Munne-Bosch 2005; Ramel et al. 2012a). The MEP pathway activity is tightly linked with photosynthetic electron transport (Dani et al. 2014a), which is the principal source of ROS in chloroplasts. The MEP pathway produces many hormones (cytokinins and abscisic acid (ABA)), volatile isoprenoids (isoprene and monoterpenes) and pigments (carotenoids). Functions of cytokinins, stored monoterpenes and carotenoids are less ambiguous than those of volatile isoprenoids in plants, as discussed in the succeeding sections. The capacity of living systems to synthesize and emit MEP-derived volatile isoprenoids has evolved under distinct selection pressures at different levels of biological complexity (Dani et al. 2015). Here, we broaden our focus from isoprene to all major products of the MEP pathway. Using the leaf as a model plant tissue, we start with a brief overview of the role of ROS in causing chloroplast genome damage and accelerating senescence in leaves. We highlight the antioxidant activity of isoprenoids and then place the metabolic trade-offs within the MEP pathway in the context of leaf ontogeny. We build an argument for common selection pressures on the MEP pathway to propose that cytokinins, volatile isoprenoids and carotenoids have evolved functional synergism in delaying plant senescence. Metabolic damage is an important feature of biological ageing. It involves deterioration of integrity and functions of macromolecules such as DNA, proteins and membrane lipids (Berlett & Stadtman, 1997; Campisi and Vijg 2009). ROS and reactive nitrogen species (RNS) act as signaling molecules at low/sublethal concentrations to recalibrate gene expression and trigger plant defense response (Goulde et al. 2003; Mittler et al. 2011). However, ROS levels in plastids and mitochondria could easily exceed tolerance thresholds causing DNA strand breakage, damage to protein synthesis machinery, membrane leakage and ultimately cell lysis (van Bresegem & Dat 2006). A high concentration of ROS (oxidative stress) triggers increased production of nitric oxide (NO) and leads to the formation of RNS-ROS complexes that could initiate the chain of events leading to programmed cell death (Wang et al. 2013). While photosystems suffer the most under excessive oxidative stress, the organelle genomes also are vulnerable to ROS-induced impairment of DNA repair mechanisms (Fig. 1a,b). The chloroplast genome (plastome) codes for critical enzymes involved in photosynthesis. Despite the harsh operating conditions within the chloroplasts, the circular genome is retained and is seen as an evolutionary redox sensor (Wright et al. 2009) and a marker of various stages of developmental (not induced) senescence. The number of plastomes per leaf mesophyll cell increases as a leaf undergoes maturation and then declines to reach a constant number after the leaf becomes fully mature (Oldenburg & Bendich 2004). The number of plastomes per cell remains stable through leaf senescence, but the quality of plastomes decline with age of the leaf (Fig. 1b; Rowan et al. 2009). Such a decline, due to fragmentation, mutations and loss of function of plastomes, is attributed to the cumulative oxidative damage suffered over the entire life span of the leaf tissue (Fig. 1c; Oldenburg et al. 2014). Experiments involving Arabidopsis mutants suggest that plastome instability because of ROS damage leads to inhibition of DNA repair proteins and to declined production and malfunction of critical proteins encoded by the plastome. This drives further ROS production and a ‘run away’ degradation of cells (Lepage et al. 2013). Thus, the loss of plastome integrity caused by ROS is central to the process of cell death and leads to tissue senescence in leaves. Among all volatile isoprenoids, the capacity to emit isoprene shows an erratic evolutionary history in higher plants and, so far, no substantial functional basis has come forth that gives an evolutionary explanation for the large loss of carbon by plants in the form of isoprene (Dani et al. 2014b). Laboratory work shows a role for isoprene in mitigating oxidative stress (Loreto & Velikova 2001), heat stress (Singsaas et al. 1997; Behnke et al. 2007; Velikova et al. 2011) and high light stress during sun flecks (Behnke et al. 2010; 2013). However, whether isoprene and non-stored monoterpenes play a similar role in field conditions, protecting emitting plants from stresses is an unsettled debate. Based on experiments in constitutive isoprenoid emitting plants, and in transgenic isoprene emitting tobacco, it was proposed that isoprene quenches ozone-induced oxidative stress and protects leaves from 1O2 (Loreto & Velikova 2001; Affek & Yakir 2002; Loreto et al. 2004; Velikova et al. 2004; Vickers et al. 2009a; 2009b). Volatile isoprenoids could react with both RNS and ROS within plastids to prevent reactive species from building to lethal levels and to stop or delay hypersensitive responses and cell death processes (Velikova et al. 2005). Overall, isoprenoids seem to play an important role in combating all types of oxidative free radicals. The antioxidant role of de novo volatile isoprenoids likely supplements the activity of carotenoids, which are important accessory pigments in the photosystems (Fig. 1). Carotenoids play a recognized role in protecting photosystems from oxidative stress (Ramel et al. 2012a, 2012b), maintaining plastome integrity and indirectly prolonging leaf life span. Even when volatiles are stored in specialized structures and not emitted constitutively, they may continue to have the function of extending tissue life span in plants. For example, volatile isoprenoids stored in leaves and barks, and released in large amounts following fungal inoculations or insect attacks, induce a ROS-mediated hypersensitive response (a type of localized PCD) to control the spread of infection (Gershenzon & Dudareva 2007; Coll 2011). While it is counterintuitive to think that isoprenoids and ROS act in concert to cause PCD, it is clear that stored volatiles prevent further damage (minimize costs) by allowing localized cell death and thus prolong longevity of the surrounding tissues (maximize benefits). Stored volatiles in scented floral petals are another example. Floral petals are essentially modified leaves both genetically and physiologically (Pelaz et al. 2001), and in scented flowers, petals emit wide range of monoterpenes. The primary role of floral monoterpenes is to attract pollinators, and in scented flowers, the emission of volatiles continues until pollination occurs (Muhlemann et al. 2014). Post-pollination floral senescence and the concurrent decline of monoterpene emission allows to (a) maximize new floral numbers and increase reproductive fitness through resource recycling; (b) avoid futile pollinator visits to pollinated flowers and increase pollinator reward for visiting un-pollinated flowers; and (c) avoid seed predation (reviewed in Muhlemann et al. 2014). Excessive ROS activity in floral petals could provide a biochemical and physiological basis to floral senescence (Rogers 2012). As in leaves, volatile isoprenoids could reduce ROS load in floral petals, indirectly slowing down floral ageing. In other words, high rates of monoterpene emission could also play a role in keeping the flower healthy (attractive) until pollinators visit and complete pollination. Cytokinins are a group of ubiquitous phytohormones synthesized by the MEP pathway in all types of plastids (e.g. amyloplasts, chloroplasts and chromoplasts) (Fig. 2). Some cytokinins are synthesized in plant roots and then delivered to other plant parts through the stream of xylem sap (van der Werf & Nagel 1996). However, significant proportions of cytokinins in young developing leaves are synthesized in situ (Singh et al. 1992). Cytokinins are involved in regulating cell cycle, cell division and tissue senescence (reviewed in Zwack & Rashotte 2013). Cytokinins promote cell division and proliferation during the early development of leaves, and, as the leaf matures, slow down cell division and maintain existing cells (i.e. active versus quiescent states of cell metabolism; Bendich 2010; Schaller et al. 2014). The mechanism of cytokinin action involves regulation of genes coding for cytokinin response factors (CRFs), DNA binding type-A response regulators (ARRs) and chloroplast-localized proteins (reviewed in Brenner et al. 2012). Tissue specific induction of cytokinin synthesis under a senescence-specific promoter prolonged the leaf life spans in transgenic tobacco, thus proving that high concentration of cytokinins in leaves is important in delaying senescence (Gan & Amasino 1995). Cytokinins delay leaf senescence by upregulating oxidative stress response genes to prevent chlorophyll decay and to maintain chloroplast integrity (Zavaleta-Mancera et al. 2007). Terpenoid phytoalexins and salicylic acid are also implicated in cytokinin-induced plant defense response to pathogen attacks (Jiang et al. 2013). As seen in leaves, overproduction of cytokinins increases corolla life span (Chang et al. 2003) and delays floral senescence (Zubko et al. 2002). However, high concentrations of cytokinins could even accelerate senescence, which is not observed in natural systems (Carimi et al. 2005; Novák et al. 2013). We visualize the MEP pathway as a system of two modules (Fig. 2, Dani et al. 2015). Modules are segments (may not always be sequential) of any pathway involving groups of genes that are under common regulation in a coordinated manner. Such modules comprise functionally related genes, often co-expressed with shared structural domains (Dani et al. 2015). In the MEP pathway, the sequence of reactions classified as terpenoid backbone biosynthesis constitute module 1 (M00096, C5 isoprenoid biosynthesis and non-mevalonate pathway Kyoto Encyclopedia of Genes and Genomes (KEGG) database; Kanehisa et al. 2014). Isoprenoid-type cytokinins, isoprene and monoterpenes acting in isoprene-like fashion when isoprene is not emitted (monoterpenes not stored in permanent pools) are part of module 1. All these end-products are made in early steps of the MEP pathway, with close interaction with the common precursor dimethylallyl pyrophosphate (DMAPP). The sequence of reactions classified as beta-carotene biosynthesis constitute module 2 of the MEP pathway (M00097, KEGG database; Kanehisa et al. 2014). Carotenoid biosynthesis has a sub-module of ABA synthesis (M00372), but we restrict ourselves to two modules for simplicity. Stored monoterpenes, constitutive monoterpenes (emitted by plants that also emit isoprene and replace monoterpenes with isoprene at leaf maturity (Brilli et al. 2009), carotenoids, chlorophyll phytyl chains and ABA constitute module 2. Junctures of metabolic trade-offs within or between modules of a biochemical pathway (i.e. points at which competition for a substrate occurs between different enzymes) are useful reference points to reveal selection pressures and functional relationships between end-products from that pathway. We expect that intermediate metabolites from the same module are regulated the same way, but end-products undergo trade-off regulation. Within the MEP pathway, as examples, we consider the relationship between (a) cytokinins and isoprene (within module 1); (b) isoprene and monoterpenes; and (c) cytokinins and monoterpenes (between modules 1 and 2). Competition for a common substrate in the synthesis of cytokinins and isoprene appears leading to contrasting changes in their de novo synthesis rates through the developmental phases of a leaf, with isoprene emission raising when in situ cytokinin production starts dropping in leaves (Fig. 3a). However, few changes in isoprene and cytokinins in leaves. we that high of isoprene for 2 and high for by and 1 isoprene emission over de novo cytokinin and monoterpene Despite such metabolic trade-offs within the MEP pathway, we that common evolutionary pressures have to functional synergism between cytokinins, volatile isoprenoids and apparent when we at the of these end-products within leaves of chloroplasts. in mature leaves, the pool of cytokinins is high because of a large from the roots through xylem (van der Werf & Nagel & van 2010), and high cytokinin is associated with high rates of volatile (Fig. In plants emitting both isoprene and monoterpenes, monoterpenes are in young developing leaves (Brilli et al. 2009), and in such monoterpenes and cytokinin a similar from to senescence (Fig. 3a). The functional synergism between cytokinins and volatile isoprenoids becomes even in plants under stresses. levels under stress with a in et al. 2013). of cytokinins by a promoter in transgenic plants that a stable cytokinin pool plant cell function under et al. et al. et al. 2013). or increased isoprenoid emission rates under observed in is proposed to be involved in protecting photosystems against and associated heat stress & Loreto Dani et al. we at carotenoids, of metabolic trade-offs within the MEP pathway becomes than discussed A trade-off between isoprene and carotenoids is seen during the early development of a leaf (Fig. et al. and it is not by competition between enzymes for the same substrate (Fig. 2). It is not clear a trade-off occurs under these However, a trade-off could be isoprene could the antioxidant function of carotenoids at a different of the life cycle of a isoprene is the for de novo carbon through the MEP pathway in et al. 2014). In isoprene transgenic the carbon isoprene is to synthesis of these two products of the MEP pathway et al. 2014). in constitutive monoterpene emission of and isoprene emission of monoterpenes) are similar et al. 1997; et al. and as discussed could their the trade-off between isoprene and carotenoids, there is no trade-off between constitutive monoterpenes and carotenoids in plants that emit monoterpenes. monoterpene and synthesis are in and decline in senescing leaves of & et al. 2009). The of trade-off between constitutive monoterpenes of and carotenoids is likely by the cycle of leaf senescence in plants. The of carotenoids in leaves remains high during the mature of a leaf, when volatile isoprenoid emission is also and thus an functional synergism between volatile isoprenoids and carotenoids is further A of the be that in plants and in all the trade-offs at biochemical within a pathway at within modules or between close modules of a may not functional between end-products from that pathway, because of and of metabolites within plants (e.g. transport of cytokinins and metabolic transport is a natural of metabolic between which is also to plant isoprenoid metabolism the function of acid pathway. In our the metabolic between and MEP pathway is not in but it becomes important under stress et al. 2003; et al. 2007). and senescence in plants are that often ROS are key factors senescence, and photosynthetic electron transport within chloroplasts is a major source of of chloroplast genomes by ROS activity is a key of leaf senescence. end-products of the MEP pathway volatile isoprenoids, carotenoids and are synthesized de novo in chloroplasts of leaves. The of these products important points in the life cycle of a We propose that relationships between the synthesis and in concentration of these metabolites beyond and they are or indirectly (i.e. by quenching of involved in regulating senescence. We propose that leaves not enter senescence as long as their cytokinin synthesized de novo from and remains and volatile isoprenoid emission remains emission is a feature of chloroplasts of plants, cytokinins and monoterpenes are synthesized in all types of plastids, in all plants. of isoprene emission with in life of leaves, Dani et al. Loreto & may suggest that isoprene emission is a on the MEP pathway, essentially the of cytokinins and carotenoids in delaying senescence. However, antioxidant and activity of isoprene and non-stored monoterpenes, we maintain that volatile isoprenoids carotenoids in quenching ROS and maintain plastome and membrane integrity to delay senescence. the among cytokinins, volatile isoprenoids and carotenoids in regulating leaf senescence. The relationship between cytokinin synthesis and isoprenoid emission is and it could many and of senescence in photosynthetic The between cytokinins and isoprenoids under stress is because leaves isoprenoid emission rates in emitting under et al. Dani et al. that overproduction of cytokinins not increase the leaf life span in transgenic et al. de novo cytokinin synthesis in leaves could play a significant role in leaf senescence and under on flowers suggest that the relationship between longevity of flowers and their volatile is ambiguous at et al. 2001; et al. 2011). not the longevity of flowers when they are and to their plants. The between cytokinin transport, metabolism and its relationship with monoterpene emission in floral petals remains and be a senescence of petals, also leads to in monoterpenes and other floral volatiles et al. 2005). It be to the between and senescence in leaves and petals in the context of monoterpene We not the functional of interaction the relationship in leaves & Loreto 2006). (a) proposed synergism between cytokinins and isoprene (b) in cytokinins and ABA levels through leaf (Fig. and (c) that the observed between isoprene and ABA in leaves, the of interaction between isoprene and ABA could be during natural senescence. we need to the of damage, and when it occurs due to prolonged oxidative stress in plants. the integrity of chloroplast DNA under oxidative stress in volatile isoprenoid emitting and plants the that the of volatile isoprenoids organelle genomes from damage (Fig. a of plants the same small but significant in and floral life spans need to be for changes in key metabolites of the MEP pathway through their life of MEP pathway products with the life and on the plants, we a between chloroplast metabolism and ageing in plants. from cytokinins, and carotenoids synthesis stored monoterpene and isoprene emission in different plant species and at different developmental stages or in are on The is not for the or of any by the than should be to the for the