Ethylene Exerts Species-Specific and Age-Dependent Control of Photosynthesis

The volatile plant hormone ethylene plays a regulatory role in many developmental processes and in biotic and abiotic stress responses. One of the underexplored actions of ethylene is its regulation of photosynthesis and associated components such as stomatal conductance, chlorophyll content, light reactions, carboxylation events, carbohydrate partitioning, and age-related senescence. In this Update, we summarize the current knowledge concerning the regulation of photosynthesis, focusing on the model species Arabidopsis (Arabidopsis thaliana). We describe how ethylene directs photosynthesis in juvenile nonsenescing leaves and mature senescing leaves. Furthermore, we extend these insights to other types of photosynthesis (C4 and Crassulacean acid metabolism) and highlight the species-specific effects of the physiological action of ethylene on this complex metabolic pathway. The volatile plant hormone ethylene (C2H4) is involved in many cellular and developmental processes, such as germination, root and hypocotyl development, climacteric fruit ripening, and senescence (Abeles et al., 1992; Van de Poel et al., 2015; Wen, 2015), as well as in the response to biotic and abiotic stress (Bari and Jones, 2009; Kazan, 2015). Photosynthesis is one of the least referenced processes regulated by ethylene, which plays a role in all three carbon fixation pathways used by plants: C3, C4, and Crassulacean acid metabolism (CAM; Box 1). Photosynthesis is a complex autotrophic process mediated by the interplay of factors such as light quantity and quality, atmospheric CO2 concentration, stomatal aperture, chlorophyll content, light-harvesting complex efficiency, sugar feedback, water availability, nutrient status, and hormonal cues. The role of ethylene in controlling photosynthesis was first described by Kays and Pallas (1980), who found that the hormone reduces photosynthesis in peanut (Arachis hypogaea). This hormonal control seems to be an ancient response in plants, because ethylene down-regulates photosynthesis in the charophyte green alga Spirogyra pratensis, suggesting that the regulation of photosynthesis by ethylene is likely conserved and predates the colonization of nonaquatic habits by plants (Ju et al., 2015; Van de Poel et al., 2016). The effect that ethylene exerts on photosynthesis depends on leaf age (Fig. 1). Ethylene directly controls photosynthesis in juvenile nonsenescing leaves and acts indirectly in mature leaves by promoting leaf senescence. In this Update, we describe the effect of ethylene on young nonsenescing leaves, focusing on the elements that influence plant photosynthesis (chlorophyll content, stomatal conductance, light dissipation, carbon fixation, and carbohydrate partitioning). We also briefly highlight the role of ethylene in leaf senescence and refer the reader to a more specialized review on this topic by Kim et al. (2015). Overview of the regulatory effects of ethylene on plant photosynthesis and its features for juvenile/nonsenescing and mature/senescing leaves. Because the regulation of photosynthesis is species specific, this general scheme does not apply to every species but merely reflects the general mode of action for the inhibitory effect of ethylene on photosynthesis as found in Arabidopsis. ABA, Abscisic acid; CTR1, CONSTITUTIVE TRIPLE RESPONSE1; EIN2, ETHYLENE INSENSITIVE 2; EIN3, ETHYLENE INSENSITIVE 3; EIL, EIN3-LIKE; ERFs, ethylene response factors; H2O2, hydrogen peroxide; PAG, photosynthetically active genes; ROS, reactive oxygen species; SAG, senescence-associated gene. The molecular regulation of photosynthesis by ethylene has been studied using ethylene-related mutants in Arabidopsis and other model plants. Box 2 highlights the steps of the ethylene signaling pathway. Grbic and Bleecker (1995) initially showed that juvenile nonsenescing leaves of the Arabidopsis ethylene-insensitive mutant ethylene resistant1 (etr1-1) possess lower chlorophyll contents, Rubisco activity, and expression of photosynthetically active genes such as CAB (chlorophyll a/b-binding protein) and Rubisco small subunit, suggesting a role for ethylene in controlling photosynthesis in nonsenescing leaves. These results were further corroborated by Tholen et al. (2004, 2007, 2008), who showed that ethylene-insensitive mutants of Arabidopsis (etr1-1 and ethylene insensitive2 [ein2]) and ethylene-insensitive transgenic tobacco (Nicotiana tabacum) plants carrying the dominant mutant allele of Arabidopsis etr1-1 have a lower whole-plant and leaf photosynthetic capacity, especially under saturating light conditions. These studies suggest that basal ethylene perception is essential for achieving normal photosynthetic capacity in Arabidopsis leaves. In addition, transient ethylene treatment of nonsenescing leaves reduces chlorophyll content (Zacarias and Reid, 1990) and down-regulates CAB expression in Arabidopsis (Grbic and Bleecker, 1995), suggesting that excessive ethylene inhibits photosynthesis in juvenile leaves. To our knowledge, no actual photosynthesis measurements from nonsenescing leaves of Arabidopsis exposed to ethylene are currently available. The control of photosynthesis by ethylene also affects plant biomass production by influencing final rosette size. Arabidopsis ethylene-insensitive mutants (etr1-1, ein2, and ein3) have larger rosettes compared with wild-type plants (Bleecker et al., 1988; Guzmán and Ecker, 1990; Ecker, 1995; Alonso et al., 1999). However, this discrepancy is probably manifested during later stages of plant development, when ethylene controls photosynthesis by stimulating leaf senescence (Fig. 1). Ethylene-insensitive mutants show a delayed onset of senescence, extending their growth period, resulting in larger rosettes compared with wild-type plants (Grbic and Bleecker, 1995; Hua et al., 1995; Alonso et al., 1999; Tholen et al., 2004). Juvenile nonsenescing 3-week-old etr1-1 mutants grown in well-ventilated conditions do not have a larger total leaf area and relative growth rate compared with wild-type plants during vegetative development despite their reduction in photosynthesis, confirming that delayed senescence in these ethylene-insensitive mutants likely controls rosette size (Tholen et al., 2004). Ethylene can stimulate the degradation of chlorophyll during fruit ripening and leaf senescence (Burg and Burg, 1965; Abeles et al., 1992). In nonsenescing developing leaves, ethylene-insensitive mutants (e.g. etr1-1) also have reduced chlorophyll content in Arabidopsis (Zacarias and Reid, 1990; Grbic and Bleecker, 1995) and protoplasts of etr1-1 have reduced chlorophyll fluorescence (Kim et al., 2017), suggesting that basal ethylene levels are required to ensure normal chlorophyll content. The opposite is true for mature leaves prone to senescence, where chlorophyll content is found to be higher in ethylene-insensitive mutants (etr1-1 and ein2-1) of Arabidopsis (Grbic and Bleecker, 1995; Oh et al., 1997), tobacco (Yang et al., 2008), and tomato (Solanum lycopersicum; Monteiro et al., 2011). Chlorophyll content is also higher in mature leaves of ACC oxidase (ACO) antisense lines of tomato (Picton et al., 1993; John et al., 1995; Jensen and Veierskov, 1998) and ACC synthase (ACS) antisense lines of maize (Zea mays; Young et al., 2004). These findings suggest that ethylene is involved in establishing normal chlorophyll content in nonsenescing leaves and promotes chlorosis through chlorophyll degradation in mature leaves (Fig. 1). Photosynthesis is intimately linked with stomatal conductance, which mediates CO2 uptake and transpiration (Males and Griffiths, 2017; Matthews et al., 2017; Vialet-Chabrand et al., 2017). Ethylene treatment by ethylene gas, ACC, or ethephon induces stomatal closure in Arabidopsis (Desikan et al., 2006). The stomata of Arabidopsis ethylene-insensitive mutants (etr1-1) show reduced conductance compared with wild-type plants (Tholen et al., 2004). These observations suggest that ethylene negatively influences stomatal conductance in Arabidopsis. It must be noted that the etr1-1 mutant also has smaller stomata (Tanaka et al., 2005), indicating that ethylene influences stomatal development. Indeed, Arabidopsis plants continuously grown in ethylene, as well as the constitutive ethylene response mutant ctr1-1, possess more stomata (Kieber et al., 1993), yet it is unknown if these plants also display higher CO2 uptake or net photosynthesis. It is not clear if altered ethylene levels play a primary role in stomatal conductance or have a secondary effect through the cross talk with ABA (Wilkinson and Davies, 2010; Murata et al., 2015). In Arabidopsis, external ethylene, or constitutive internal ethylene production by the eto1-1 mutation, inhibits ABA-induced stomatal closure (Tanaka et al., 2005; Watkins et al., 2014). The ethylene signaling mutants (etr1-1 and ein3-1) and the ethylene perception inhibitor 1-methylcyclopropane do not affect ABA-induced stomatal closure (Tanaka et al., 2005). The inhibition of ABA-induced stomatal closure also is observed in wheat (Triticum aestivum), but older leaves are more sensitive to ethylene while younger leaves are more sensitive to ABA (Chen et al., 2013). The inhibitory action of ethylene on ABA-induced stomatal closure is regulated mainly by hydrogen peroxide signaling (Desikan et al., 2005, 2006; Shi et al., 2015). Ethylene stimulates flavonol production in the guard cells, which subsequently suppresses reactive oxygen species accumulation and consequently reduces ABA-induced stomatal closure (Fig. 1; Watkins et al., 2014, 2017). The capture of light energy by PSI and PSII is necessary to accommodate electron transport and the reduction of CO2 (the so-called dark reactions of photosynthesis) in plant chloroplasts. Wullschleger et al. (1992) reexamined the light-response and CO2-response curves of soybean (Glycine max) reported by Taylor and Gunderson (1988) and found a 30% decline in the electron transport capacity following a 4-h exposure to ethylene. In line with these results, transient overexpression of a specific ethylene-responsive factor (CitERF13) in tobacco leaves compromised photosynthetic rates. Significant declines of the maximum quantum efficiency as well as the effective quantum efficiency of both PSI and PSII occurred 3 and 2 d, respectively, after infiltration with Agrobacterium tumefaciens carrying CitERF13 (Xie et al., 2017). In addition, pulse-amplitude modulation fluorimetry-based chlorophyll fluorescence analyses by Kim et al. (2017) reveal that ethylene-insensitive mutants of Arabidopsis (etr1-1) display lower PSII activity compared with wild-type plants. These results are further corroborated by augmented expression of the cellular energy stress sensor AKIN10 (ARABIDOPSIS KINASE10) in ethylene-insensitive mutants. AKIN10 is an isoform of the evolutionarily conserved energy sensor SNF1-RELATED PROTEIN KINASE (SnRK1) and has the ability to modulate the ratio of catabolism to anabolism to sustain cellular viability (Broeckx et al., 2016). It must be noted that a second mutation in the Arabidopsis etr1-1 mutant produces a premature stop codon in ARC3 (ACCUMULATION AND REPLICATION3), which is responsible for the abnormally large chloroplasts observed in etr1-1 but not ein2 and ein3 mutants (Cho et al., 2012). ARC3 plays a role in chloroplast division (Maple et al., 2007), and the arc3 mutant displays abnormal chloroplasts in mesophyll protoplasts independent of the etr1-1 mutation (Cho et al., 2012). Given this important secondary mutation (arc3) in the etr1-1 mutant line commonly used in ethylene research, it is advised to seek additional confirmatory lines of evidence, especially when working on photosynthesis. Nonetheless, ethylene signaling mutants outcrossed from the arc3 secondary mutation (etr1-1sg) also are found to have a lower maximum quantum efficiency, higher chlorophyll fluorescence lifetime of PSII, and, consequently, a lower quantum yield of PSII (Kim et al., 2017). These findings suggest that normal ethylene sensitivity is required for optimal photochemical efficiency of PSII independent of an altered chloroplast structure caused by the secondary arc3 mutation (Kim et al., 2017). Besides photochemical quenching, ethylene also has been found to influence nonphotochemical quenching properties by the intervention of the xanthophyll cycle mechanism. Chen and Gallie (2015) demonstrated that Arabidopsis eto1-1 mutants, with increased ethylene production, are affected in their ability to convert violaxanthin to zeaxanthin by impairing violaxanthin deepoxidase activity. As a consequence, these plants are particularly prone to elevated reactive oxygen species production and photosensitivity. A study by Grbic and Bleecker (1995) showed that young leaves of Arabidopsis ethylene-insensitive mutants (etr1-1) have reduced Rubisco activity while older leaves have higher Rubisco activity compared with wild-type plants. Using ethylene-insensitive transgenic tobacco plants, Tholen et al. (2007) also revealed decreases in Rubisco transcripts and protein content of about 76% and 42%, respectively, in comparison with wild-type plants. A high degree of down-regulation of Rubisco occurs for both ethylene-insensitive tobacco and Arabidopsis seedlings growing on a Glc-enriched medium (Zhou et al., 1998). Further investigations into the decreased rates of photosynthesis in tobacco after transient overexpression of CitERF13 reveal that the maximum rate of Rubisco carboxylase activity is indeed affected (Xie et al., 2017). We can conclude from these reports that normal ethylene sensitivity is required to achieve maximal Rubisco activity in nonsenescing leaves. As Rubisco is inefficient due to its typical error-prone catalytic properties (Bracher et al., 2017), ethylene also is likely to influence the protein abundance and/or activity of Rubisco activases (RCAs), adding an additional layer of complexity to mediating the carboxylation process. RCAs are well known as molecular chaperones for Rubisco to deal with its dead-end inhibited complexes due to its high affinity for its substrate ribulose 1,5-bisphosphate and similar sugars when the active site has been left unprimed with CO2 and Mg2+ cofactors (Parry et al., 2008). Khan (2005) reported an increased activity of carbonic anhydrase (CA) in mustard (Brassica juncea) upon treatment with the ethylene-releasing growth regulator ethephon. Besides participating in the regulatory network to control chloroplast pH and protect stroma enzymes from denaturation during severe and sudden changes in light conditions, CA can elevate the concentration of CO2 in close proximity to Rubisco and, as such, limit the proportion of photorespiration (Badger, 2003). CA also plays a role in C3 but even more critically in C4 and CAM photosynthesis, providing sufficient bicarbonate to fuel phosphoenolpyruvate carboxylase (PEPC; DiMario et al., 2017). Work on the effect of ethylene on carbon fixation in C4 and CAM plants is limited. Young et al. (2004) reported that an acs6 mutation in maize (C4) results in lower ethylene production, leading to higher Rubisco content and higher CO2 assimilation for both young and mature leaves. These results suggest that basal levels of ethylene suppress maximal carbon fixation in maize. We have not found other studies that report the effect of ethylene on Rubisco, CA, or PEPC in other C4 or CAM plants, making it difficult to draw firm conclusions about the impact of ethylene on C4 and CAM carboxylation (Fig. 2). Overview of the three modes of photosynthesis (C3, C4, and CAM) in plants, indicating the importance of Rubisco and PEPC(phosphoenolpyruvate carboxylase) as main enzymes involved in carbon fixation. Overview of the plant ethylene signal transduction pathway. Ethylene binds the ethylene receptors (ETR) at the endoplasmic reticulum (ER), resulting in autophosphorylation of the receptor and inactivation of the downstream kinase CTR1. In the absence of ethylene, CTR1 phosphorylates the ER-localized EIN2, directing it for proteasomal degradation by the ETPs(EIN2 TARGETING PROTEINS). When ethylene is present, the EIN2 C-terminal part (EIN2-C) is not phosphorylated and gets cleaved and migrates to the nucleus, where it activates the master transcription factor EIN3 and EILs. In the absence of ethylene, EIN3 and EILs are targeted for proteasomal degradation by the ETHYLENE BINDING FACTOR (EBF). EIN3 and EILs, in turn, activate ethylene response factors (ERFs) or ethylene-responsive genes. The EIN2-C also can interact with the mRNA of selected transcripts, including the 5′ untranslated region of the EBF mRNA, to inhibit translation. Following diurnal synthesis, triose phosphates need to be continuously removed from the Calvin cycle by either being deployed for starch formation in the chloroplast or exported to the cytosol to fuel Suc synthesis to replenish nonphotosynthetic parts of the plant. Suc transport is for achieving optimal carbohydrate in a plant that is but also to To the carbohydrate and energy status, plants an network of and such as and and et al., 2014). It is that ethylene also in carbohydrate partitioning, as have been in the complex that sugar and hormone signaling (Fig. 1). The and ethylene signaling a (Fig. 1; et al., and 2003). Ethylene-insensitive plants ein2, and ein3) are to to the while constitutive ethylene or ethylene mutants or ACC results in decreased sugar sensitivity (Zhou et al., and et al., 2003). also cross with ethylene by stimulating EIN3 which reduces ethylene a that sensitivity et al., 2003). in the increased production after ethylene treatment is by the abundance of and which to the et al., et al. (2017) also reported altered expression of the genes and upon ethylene treatment in the In addition, ethylene was to the to the period, a response that can be by Suc et al., 2017). These findings that ethylene and the of Suc from photosynthesis can cross talk to influence and control plant photosynthetic capacity et al., 2017). Besides influencing photosynthesis in nonsenescing leaves, ethylene also inhibits photosynthesis through the and of senescence in mature leaves (Fig. 1; Kim et al., 2015). It has been that external ethylene stimulates leaf senescence (Bleecker et al., 1988; and Reid, while ethylene-insensitive mutants (etr1-1, and in Arabidopsis and in show reduced rates of senescence (Bleecker et al., 1988; et al., Grbic and Bleecker, 1995; Hua et al., 1995; Oh et al., et al., Kim et al., 2014). etr1-1 and mutants have increased ethylene production compared with wild-type plants and Ecker, 1990; et al., yet their ethylene the additional ethylene, resulting in a in leaf senescence. ethylene-insensitive mutants age and show senescence, suggesting that ethylene controls both the rate and of leaf senescence (Grbic and Bleecker, senescence and declines in chlorophyll content and photosynthesis also have been observed in antisense plants for of tomato (Picton et al., 1993; John et al., 1995; Jensen and Veierskov, 1998) and acs6 mutants of the C4 plant maize et al., but not for mutants of Arabidopsis et al., Arabidopsis plants continuously exposed to ethylene do not display an rate of senescence (Kieber et al., but have smaller similar to and constitutive mutants and Guzmán and Ecker, 1990; et al., 1993; et al., plants also show altered leaf development but no of senescence et al., In we conclude that a exposure to ethylene stimulates leaf senescence in mature leaves while exposure altered leaf development. A transient ethylene treatment of mature leaves stimulates senescence by the expression of (Grbic and Bleecker, 1995; et al., et al., 1). important have been to play a role in cellular processes, such as chlorophyll sugar and 2015). The master transcription factor EIN3 has been as an important in controlling et al., 2013). It can activate the expression of and important transcription factors controlling leaf senescence et al., Kim et al., 2014). Furthermore, EIN3 binds to the of the and inhibits its expression during leaf development et al., 2013). In turn, inhibits the expression of the transcription factor (Kim et al., et al., a and regulation of senescence by ethylene through the action of EIN3 (Fig. 1). review that the effects of ethylene on of photosynthesis for both juvenile nonsenescing and mature senescing leaves be physiological with ethylene have revealed a species-specific reduction in photosynthesis in plants important ethylene has no effect on photosynthesis in other species Pallas and et al., Taylor and In the mustard species ethylene was found to stimulate photosynthesis et al., 2011). Besides the in ethylene also photosynthesis in other such as green and green ethylene has no effect on and Pallas and et al., Taylor and et al., When these it be noted that in growth conditions, of the and/or developmental it to draw firm but the general is that ethylene inhibits photosynthesis in nonsenescing leaves or has no effect at all on photosynthesis, with the of Furthermore, studies on tomato and (Solanum have that a ethylene treatment and its in a response of photosynthesis, with of and photosynthesis compared with the control et al., 1992; et al., 2003). These studies into the regulation of photosynthesis by ethylene in tomato and and stress the importance of the of after the In addition, et al. and and that the reduction of photosynthesis in tomato and is caused mainly by the reduced light perception of leaves as a of the response by ethylene. The of the leaves the light is important that influences net photosynthesis. and the that our knowledge concerning the influences of ethylene on photosynthesis is mainly from C3 C3 plants are with the of maize and green C4 plants are C4 photosynthesis in maize seems by ethylene treatment and et al., Taylor and while mutants of of maize show reduced ethylene resulting in higher chlorophyll and Rubisco and higher photosynthesis rates et al., 2004). These suggest that levels of basal ethylene to suppress maximal photosynthetic activity in maize. To our knowledge, are no reports that describe the effect of ethylene on CAM one reports ethylene with the plant but in the C3 mode et al., 2004). As no in either acid or PEPC transcripts be observed after treatment with ethylene, et al. (2004) that ethylene was not involved in signaling of the The process of photosynthesis plants with energy and and other on by the production of have been in our of the process of photosynthesis and its yet its hormonal regulation by ethylene This highlights that ethylene exerts species-specific regulation on plant photosynthesis, where it reduces photosynthesis in species and has no effect in other It must be noted that ethylene exerts a action in which it reduces photosynthesis in young photosynthetically active leaves and inhibits photosynthesis in older leaves by the of leaf senescence. We have the findings on how ethylene in processes, such as chlorophyll light reactions, stomatal conductance, carboxylation events, carbohydrate partitioning, and leaf senescence. The of how ethylene controls these processes and more are to a how ethylene controls one of the processes in plants. In the this knowledge be into that We the for their