Keywords
Photorespiration, high temperature, high-light, alternative electron acceptors, cowpea, , heat stress, net CO2 assimilation, PSII efficiency
INTRODUCTION
Heat stress is a major environmental challenge for plants, and it is well established that, in a wide range of species, high temperature (HT) stress can lead to declines in photosynthesis (Cui, Li, Fan, Xu, & Zhang, 2006; Panigrahi, Pradhan, Panda, Panda, & Joshi, 2016; Tan, Meng, Brestic, Olsovska, & Yang, 2011; Wise, Olson, Schrader, & Sharkey, 2004). The impact of HT stress on photosynthesis is dependent on factors such as leaf developmental age, duration and previous history of exposure (Hasanuzzaman, Nahar, Alam, Roychowdhury, & Fujita, 2013), as well as other environmental factors, especially high-light (HL) stress, which often co-occur with and can be compounded by HT (Weis, 1982).
While light energy is essential for photosynthesis, excessive light can cause detrimental effects on photosynthesis, a phenomenon known as photoinhibition or photodamage (Aro, Virgin, & Andersson, 1993; Long, Humphries, & Falkowski, 1994; Nishiyama & Murata, 2014). HL stress causes reductions in photosynthesis, especially under rapidly fluctuating environmental conditions, which ultimately results in loss of crop productivity (Slattery, Walker, Weber, & Ort, 2018). Plants have evolved various mechanisms to deal with excessive light and prevent damage, including changes in leaf orientation (Gamon & Pearcy, 1989; Pastenes, Pimentel, & Lillo, 2005), chloroplast movement (Suetsugu, Higa, Gotoh, & Wada, 2016; Wada, 2013) and non-photochemical quenching (NPQ) processes that dissipate excitation energy (Ruban, 2016; Stefanov & Terashima, 2008).  There are several forms of NPQ, including the rapidly reversible “energy dependent” quenching (q E), which is activated by acidification of the thylakoid lumen (Avenson, Cruz, & Kramer, 2004; Li et al., 2004), and more slowly-reversible forms including q I, which is caused by accumulation of inactive (photodamaged) photosystem II (PSII) centers (Krause, Somersalo, Zumbusch, Weyers, & Laasch, 1990) and state transitions (q T) that can divert PSII-associated light harvesting complexes (LHCs) to PSI. Depending on its redox state, PSI can either use light energy from these LHCs for PSI photochemistry, or dissipate it as heat (Butler, 1978; Joly & Carpentier, 2007).
The importance of light for thermotolerance of photosynthesis has been noted in previous work (Buchner, Stoll, Karadar, Kranner, & Neuner, 2015; Havaux, Greppin, & Strasser, 1991; Weis, 1982). Although, there have been several studies that show that HL combined with HT accentuates photoinhibition (e.g. Chen et al., 2017; Havaux, 1992; Lu et al., 2017), there are also reports, for instance in tomatoes, where combined moderate HT and HL improved tolerance of photosynthesis compared to either treatment alone (Gerganova, Popova, Stanoeva, & Velitchkova, 2016). However, since different species have different light intensity requirements (e.g shade vs. sun adapted plants, (Hemming, 2011; C. H. Lin, McGraw, George, & Garret, 1999)), it is very likely that responses to combinations of HT and HL stress are dependent on genotype, species or acclimation during development. Nevertheless, several components of the responses may be conserved among species.
During photosynthesis, light energy captured by the light harvesting complex is used to drive electrons by linear electron flow (LEF) to store reducing power in NADPH/O2 and phosphorylation potential in ATP/ADP+Pi, which together drive the carbon assimilation reactions in the Calvin-Benson-Bassham (CBB) cycle and other cellular processes. In principle, both the light reactions and CBB cycle may be affected by HT. For example, it is well-established that HT decreases the specificity of rubisco and solubility of CO2relative to O2 (Brooks & Farquhar, 1985; Galmés, Hermida-Carrera, Laanisto, & Niinemets, 2016), thus increasing “photorespiratory pressure” or rates of photorespiration faster than the rate of rubisco carboxylation (Schuster & Monson, 1990).
In addition to this shift from assimilation to photorespiration, moderately high temperatures (35–40 °C) decrease rubisco activity, which can be attributed to the loss of rubisco activase activity, leading to progressive deactivation of rubisco by accumulation of inhibitory products such as sugar phosphates (Crafts-Brandner & Salvucci, 2000; Salvucci & Crafts-Brandner, 2004). While these studies suggest that reduction in rubisco activity is a primary deleterious effect of HT stress, it has also been proposed (Sharkey, 2005) that this effect constitutes an adaptive mechanism possibly to avoid situations where the damage by toxic products of photorespiration outweighs the advantage of higher carbon fixation rates.
A rubisco activase feedback mechanism of this type could allow for “safe” downregulation of assimilation. However, this mode of regulation should decrease the availability of sinks for the products of the light reactions. Without coordinated downregulation of the light reactions, this mode of regulation could result in the accumulation of reactive intermediates and subsequent photodamage. Indeed, it has been proposed that high rates of photorespiration under environmental stresses can serve a photoprotective role by maintaining electron acceptor sinks (Huang, Hu, & Zhang, 2015; Voss, Sunil, Scheibe, & Raghavendra, 2013). However, if rubisco is completely deactivated, rates of photorespiration would also be decreased. Thus, many times HT results in decreases in assimilation that are not fully accounted for by increased photorespiration (Sharkey, 2005), suggesting that feedback systems must operate to coordinate the capture of light in response to these changes in assimilation, as suggested by observations that HT induced decreases in assimilation in wheat were not accompanied by increased PSII photodamage (Kalituho, Pshybytko, Kabashnikova, & Jahns, 2003).
The light and assimilatory reactions are tightly co-regulated to balance the needs for efficient energy capture, while balancing energy storage into ATP and NADPH to meet the needs of downstream metabolic reactions and prevent the accumulation of reactive intermediates that can lead to photodamage (Kramer & Evans, 2011; (Walker, Kramer, Fisher, & Fu, 2020). The thylakoid proton motive force (pmf ) plays a central role in this co-regulatory network (Avenson et al., 2005; Kanazawa & Kramer, 2002). The ΔpH component of pmf causes acidification of the lumen, which activates q E (Li, Muller-Moule, Gilmore, & Niyogi, 2002; Ruban, Johnson, & Duffy, 2012) and controls electron flow at the level of the cytochromeb 6f complex, in a process known as “photosynthetic control” (Tikhonov, 2014). The pmf and its effects on downregulation are sensitive to processes that alter the influx of protons such as LEF, cyclic electron flow (CEF) and the conductivity of the thylakoid membrane to proton efflux (g H+). The latter is governed mainly by the chloroplast ATP synthase, and the partitioning ofpmf into its two components, Δψ and ΔpH. For example, decreasing CO2 level slows assimilation and thus decreases LEF and the flux of protons into the lumen. However, ATP synthase also slows, so despite the fact that proton influx is slowed, the thylakoid builds up a larger pmf , leading to activation of q Eand increased photosynthetic control (Kanazawa & Kramer, 2002). Moderate HT has been shown to modulate this co-regulatory network in tobacco by altering g H+, the rates of violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE) and the partitioning of pmf into Δψ and ΔpH (Zhang, Kramer, Cruz, Struck, & Sharkey, 2011), but it is unclear how HL interacts with these regulatory responses.
In this study, we focused on the effect of light on responses of photosynthesis to HT in cowpea (Vigna unguiculata ) seedlings having leaves of different maturity, with the aim of determining the relative effects on assimilation and light reactions and their co-regulation. We focused on cowpea because it is an agriculturally important crop that serves as an essential source of livelihood and nutrition for many in developing countries, many of which have hot climates. Characterized by quick growth and broad leaves, it is ideal for studying the interaction between HL and HT since it exhibits considerable genetic variation in tolerance to these traits (Ehlers & Hall, 1996, 1998).
Our data shows that in low light (LL), HT caused substantial decreases in CO2 assimilation and PSII activity, while HT in combination with HL led to a stimulation of PSII activity, but decreases in CO2 assimilation compared to growth temperature (GT). Further experiments revealed differences in the rates of PSII photodamage between GT+HL and HT+HL, whereas PSII activity was not enhanced in HT+HL under non-photorespiratory conditions, suggesting photorespiration and other alternative electron acceptors to be essential for tolerance of PSII to HT+HL stress.