Update on Dynamic Photosynthesis in Crops

Fluctuating Light Takes Crop Photosynthesis on aRollercoaster Ride1[OPEN]

Elias Kaiser,a,2 Alejandro Morales,b and Jeremy Harbinsona

aHorticulture and Product Physiology Group, Wageningen University, 6700 AA Wageningen, The NetherlandsbCentre for Crop Systems Analysis, Wageningen University, 6700 AK Wageningen, The Netherlands

ORCID IDs: 0000-0002-9081-9604 (E.K.); 0000-0002-6129-4570 (A.M.); 0000-0002-0607-4508 (J.H.).

The environment of the natural world in whichplants live and have evolved and within which photo-synthesis operates is one characterized by change. Thetime scales over which change occurs can range fromseconds (or less) all the way to the geological scale. Allof these changes are relevant for understanding plantsand the vegetation they create. In this update review,we will focus on how photosynthesis responds tofluctuations in irradiance with time constants up to therange of tens of minutes. Photosynthesis is a highlyregulated process, in which photochemistry as well asthe electron and proton transport processes leading tothe formation of ATP and reducing power (reducedferredoxin and NADPH) need to be coordinated withthe activity of metabolic processes (Foyer and Harbin-son, 1994). Light, temperature, the supply of the pre-dominant substrate for photosynthetic metabolism(CO2), and the demand for the products of photosyn-thetic metabolism are all factors that are involved inshort-term alterations of steady-state photosyntheticactivity. The coordinated regulation of metabolismwith the formation of the metabolic driving forces ofATP and reducing power is subject to various con-straints that limit the freedom of response of the system.Of these constraints, themost prominent are the need tolimit the rate of formation of active oxygen species bylimiting the lifetime of excited states of chlorophyll aand the potential of the driving forces for electrontransport (Foyer andHarbinson, 1994; Foyer et al., 2012;Rutherford et al., 2012; Murchie and Harbinson, 2014;Liu and Last, 2017), limiting the decrease of lumen pHto avoid damaging the oxygen evolving complex ofPSII (Krieger and Weis, 1993), and adjusting stomatal

conductance (gs) to optimize photosynthetic water-useefficiency (Lawson and Blatt, 2014).

The processes that regulate electron and protontransport, enzyme activation, and CO2 diffusion intothe chloroplast under steady-state conditions also reactin a dynamic and highly concerted manner to changesin irradiance, balancing between light use and photo-protection. This overview of the physiological controlunderlying dynamic photosynthesis is specific to the C3photosynthetic pathway. Much less is known about thedynamic regulation of the C4 and Crassulacean acidmetabolism (CAM) pathways, though given their C3

1 Much of the work leading up to this review was performed un-der the Dutch national program “Biosolar Cells.” During the elabo-ration of this review, A.M. was financed by the research program“Crop sciences: improved tolerance to heat and drought”with projectnumber 867.15.030 by the Netherlands Organisation for Scientific Re-search. J.H. would like to acknowledge financial support from the EUMarie Curie ITN “Harvest” (grant 238017).

2 Address correspondence to elias.kaiser@wur.nl.E.K., A.M., and J.H. cowrote the article. E.K. and A.M. performed

sunfleck measurements. A.M. performed data analysis of sunflecksand cloudflecks.

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heritage, we expect that they share much of the regu-lation of C3 photosynthesis. We note here that in com-parison to C3 plants, some C4 species, including maize(Zea mays), show a very slow photosynthetic inductionafter an irradiance increase (Furbank andWalker, 1985;Chen et al., 2013) and that this phenomenon deservesfurther attention.

If we grant that the regulation of photosynthesis atsteady-state is in some way optimal and represents anideal balance between light-use efficiency and photo-protection and an ideal balance between CO2 diffusioninto the leaf with the loss of water vapor from the leaf,then significance to photosynthesis under a fluctuatingirradiance is the loss of optimal regulation. The fasterthe response to change, the less is the loss of efficiency,

whether that be in terms of water use efficiency (WUE)or light use efficiency.

Since its birth one hundred years ago (Osterhout andHaas, 1918), research on the dynamics of photosyn-thesis and the limitations it produces in a fluctuatingirradiance has come a long way (Box 1). While it hasbeen apparent for some time that sunflecks occur in allkinds of canopies (e.g. Pearcy et al., 1990), research onsunfleck photosynthesis was until recently driven by itsimportance for forest understory shrubs and trees. Theecophysiological importance of sunflecks, photosyn-thetic responses, and plant growth focused on the im-portance of these responses for understory plantsgrowing in shade (Pearcy et al., 1996; Way and Pearcy,2012). Attention has more recently shifted to crop

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stands grown in full sunlight and the fact that the slowresponse of photosynthesis to sunflecks is a limitationto crop growth in the field (e.g. Lawson et al., 2012;Carmo-Silva et al., 2015). The importance of improvedphotosynthesis as a route to improving crop yields (Ortet al., 2015) has given new impetus into better under-standing the physiology and the genetics of photosyn-thetic responses to fluctuating light and improvingupon them (e.g. Kromdijk et al., 2016).

FLUCTUATING IRRADIANCE IN CANOPIES

Sunflecks

Most studies have focused on irradiance fluctuationsat the bottom of canopies or in forest understories. Inthese situations, a shade environment with little diurnalvariation prevails, and most incoming irradiance ar-rives due to transmission and scattering by leaves higherup in the canopy. Also, gaps in the canopy, which movein response to wind, allow brief but significant increasesin irradiance (Pearcy, 1990). Smith and Berry (2013)proposed a detailed classification of these fluctuations,resulting in the terms sunfleck (,8 min and peak irra-diance lower than above-canopy irradiance), sun patch(.8 min), sun gap (.60 min), and clearing (.120 min).In addition to the length of the fluctuation, classifying

a fluctuation as a sunfleck depends on the irradianceincreasing above a specific threshold during the fluc-tuation. Often, fixed thresholds are used, but their valuesvary greatly (60–300 mmol m22 s21; Pearcy, 1983, 1990;Tang et al., 1988; Roden and Pearcy, 1993; Barradas et al.,1998; Naumburg and Ellsworth, 2002). Thresholds maybe adjusted depending on canopy structure, positionwithin the canopy where measurements are taken, andangle of measurement (Pearcy, 1990; Barradas et al.,1998). An alternative approach is to use the fraction ofirradiance transmitted by the canopy instead of absoluteirradiance to calculate the threshold (Barradas et al.,1998). However, this approach requires an additionalmeasurement of irradiance above the canopy.Short-lived sunflecks with low peak irradiance are

particularly abundant in the lower layers of canopiesand forest understories. Pearcy et al. (1990) reportedthat 79% of sunflecks were #1.6 s long in a soybean(Glycine max) canopy, and the same distribution wasreported for aspen (Populus tremuloides; Roden andPearcy, 1993). Peressotti et al. (2001) reported that mostsunflecks in wheat (Triticum aestivum), maize, andsunflower (Helianthus annuus) were #1 s long. Mostsunflecks in bean (Phaseolus vulgaris) and rice (Oryzasativa) canopies were #5.0 s long (Barradas et al., 1998;Nishimura et al., 1998). These results agree with ourmeasurements in durum wheat (Triticum durum) andwhite mustard (Sinapis alba; Fig. 1).Canopy structure is assumed to affect sunfleck dis-

tribution (Pearcy, 1990), but this has so far only beensystematically tested by Peressotti et al. (2001), whocompared sunflecks in different crop canopies and

found only small differences between wheat, maize,and sunflower. Our data, on the other hand, revealedbigger differences between crops despite similar mete-orological conditions (Fig. 1): in durum wheat, 2,606sunflecks (83% of total irradiance) were detected within6 h, while only 213 (22%) were observed in whitemustard (Fig. 1A). In white mustard, sunflecks tendedto be shorter and weaker, though for both crops mostsunflecks were ,5 s long (Fig. 1B). For most sunflecks,the average irradiance increase was,350 mmol m22 s21

, and peak irradiance was always below the irradiancemeasured above the canopy (Fig. 1A). However, a largeproportion of short sunflecks may not always contrib-ute much to integrated irradiance, partly because oftheir short duration and partly because of their lowpeak irradiance (Pearcy, 1990). For example, in a soy-bean canopy, the peak irradiance in sunflecks less than1.6 s long was two to three times less than that of longersunflecks, and contributed only 6.7% of the total irra-diance, while sunflecks lasting up to 10 s contributedonly 33% of the total irradiance (Pearcy et al., 1990).

Sunflecks can also be caused by the penumbra effect(Smith et al., 1989), a “soft shadow” that occurs when alight source is partially blocked. In canopies, a pe-numbra is produced by small canopy elements thatpartially obscure the solar disc as viewed from a lowerleaf. When combined with rapid leaf movements, thepenumbra causes sunflecks on leaves that are otherwiseshaded. Due to the penumbra effect, it was estimatedthat a gap in a canopy must have an angular size greaterthan 0.5° in order for the sunfleck to reach full solar irra-diance (Pearcy, 1990). The frequent, short sunflecks dis-cussed above are probably caused by penumbra (Smithand Berry, 2013) and contribute to a substantial fraction oftotal irradiance in forest understories (Pearcy, 1990).

Due to wind-induced movements, the structure ofcanopies is not static. Wind has two effects: (1) move-ment of the whole plant or “swaying” (de Langre, 2008;Tadrist et al., 2014; Burgess et al., 2016) and (2) flutter-ing of single leaves, especially in trees (Roden andPearcy, 1993; Roden, 2003; de Langre, 2008). Plantswaying alters the spatial distribution of canopy gapsand the exposure of leaves to these gaps, adding sun-flecks and shadeflecks to the baseline irradiance thatwould occur in the absence of wind. Fluttering allowsindividual leaves to have a more uniform diurnal dis-tribution of absorbed irradiance and to maintain a highphotosynthetic induction state (Roden, 2003). Flutter-ing further increases the number of sunflecks at thebottom of the canopy (Roden and Pearcy, 1993). Leavesflutter at a wide frequency range (1–100 Hz; Roden andPearcy, 1993; Roden, 2003; de Langre, 2008), whereasplant swaying occurs at 0.1 to 10 Hz (de Langre, 2008;Burgess et al., 2016). Wind thus introduces rapid irra-diance fluctuations in the entire canopy. Without wind,sunflecks and shadeflecks can still be caused by gaps inthe canopy structure and by penumbra, but high windspeeds have been correlated with increasing irradiancefluctuations (Tang et al., 1988).

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Shadeflecks

As long as the total irradiance intercepted by a canopyremains the same, the existence of sunflecks necessitatesthe existence of shadeflecks (i.e. transient excursions be-low a baseline that is the average irradiance (Pearcy, 1990;Pearcy et al., 1990; Barradas et al., 1998; Lawson et al.,2012). It is important to distinguish between sunflecksand shadeflecks, as the dynamic responses of photosyn-thesis are different for increasing and decreasing irradi-ance and involve different potentially limiting processes(see below). A shadefleck should not be seen as a “periodbetween sunflecks,” but rather as a brief period of lowirradiance with respect to a baseline of intermediate orhigh irradiance, which tends to occur in the top andmiddle layers of a canopy.A special type of shadefleck is acloudfleck (Box 2; Knapp and Smith, 1988).

THE REGULATION OF PHOTOSYNTHESIS INFLUCTUATING IRRADIANCE

Responses and Regulation of Electron andProton Transport

The shorter term physiological responses of photo-synthesis begin with light-driven redox state and pH

changes occurring within and close to the thylakoidmembranes. Photochemistry, the primary chemicalevent of photosynthesis, provides the redox drivingforces for electron and proton transport, which result inthe feed-forward activation of metabolic processes thatproduce CO2 assimilation. Metabolism, when limiting,will down-regulate electron transport via feed-backmechanisms. This balance between feed-forward andfeed-back regulation is at the heart of photosyntheticregulation, including responses to changing irradiance.

In a leaf initially subject to a subsaturating irradiance,a sudden increase in irradiance results in an increase inthe rate of photochemistry and then an increase in therate of linear electron flow (LEF) from water to ferre-doxin within milliseconds. For every electron passingalong the LEF, three protons are translocated from thestroma into the thylakoid lumen, which changes theelectric (Dc) and pH (DpH) gradients across the thyla-koid membrane. Together, Dc and DpH constitute theproton motive force (pmf). The pmf is further modu-lated by cyclic electron flux around PSI (Strand et al.,2015; Shikanai and Yamamoto, 2017) and alternativenoncyclic electron flux (Asada, 2000; Bloom et al., 2002),making the pmf more flexible to changing metabolicdemands for ATP and NADPH (Kramer and Evans,2011) and adjustments in lumen pH resulting in

Figure 1. Sunflecks in two crop canopies. A, Irradiance fluctuations above and below a durumwheat andwhite mustard canopy,logged at 1 s resolution. B, Fraction of the total number of sunflecks as a function of sunfleck duration; calculations based on datadisplayed in A. Photosynthetically active irradiance (PAR; 400–700 nm) was logged using two LI-190R quantum sensors (Li-CorBiosciences) and an LI-1400 (Li-Cor) data logger. Data were recorded 10 cm above the ground for measurements below canopiesand just above canopies for 6 h (11:00–17:00) on two consecutive days (May 26 and 27, 2017) in Wageningen, The Netherlands(51.97 °N, 5.67 °E, 12 m above sea level). The two days were cloudless with average wind speeds of 3.5 m s21 and 4.2 m s21,respectively. In the absence of sunflecks, the irradiancemeasured below the canopywas 2.4% and 3.7%of above-canopy PAR forwhite mustard and wheat, respectively, indicating full canopy closure. To detect sunflecks, a baseline was constructed by in-terpolating PAR values in the absence of sunflecks and defining a sunfleck as the absolute change in PAR with respect to thebaseline .10 mmol m22 s21 (this was larger than the measurement error).

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regulatory responses of thylakoid electron transportand nonphotochemical quenching (NPQ). The acidifi-cation of the lumen upon increases in irradiance par-tially drives the fastest component of NPQ (Fig. 2), qE.This form of NPQ acts to reduce the lifetime of excitedsinglet states of chlorophyll a (1chl*) in PSII. When therate of PSII excitation and 1chl* formation exceeds thepotential for photochemical dissipation of 1chl* viaelectron transport (e.g. during irradiance increases), thelifetime of 1chl* in PSII tends to increase, potentiallyincreasing the rate of formation of triplet chlorophyllsin the PSII pigment bed and reaction center, resulting inthe formation of reactive singlet oxygen (Müller et al.,2001). Up-regulating NPQ activity counteracts thetendency for increased 1chl* lifetime and moderates theincrease in singlet oxygen formation (Müller et al.,2001). The protein PsbS senses the low pH in the lumen(Li et al., 2000, 2002) and may mediate conformationalchanges in trimeric light harvesting complex II (LHCII)antenna complexes that allow the light harvesting com-plex (LHC) to more efficiently dissipate excitons formed inPSII as heat (Ruban, 2016). The presence of the carotenoidzeaxanthin further amplifies qE (Niyogi et al., 1998). Zea-xanthin is formed from violaxanthin via antheraxanthin bythe enzyme violaxanthin de-epoxidase upon acidificationof the thylakoid lumen and is reconverted to viola-xanthin as lumen pH increases (Demmig-Adams, 1990).

Since after drops in irradiance NPQ relaxes onlyslowly (Fig. 2), LEF is transiently limited by an over-protected and quenched PSII, potentially limitingphotosynthesis (Zhu et al., 2004). In Arabidopsis (Ara-bidopsis thaliana), the DpH component of the pmf wasincreased in plants overexpressing K+ efflux antiporterproteins, accelerating NPQ induction and relaxationkinetics and diminishing transient reductions in LEFand CO2 assimilation upon transitions from high to lowirradiance (Armbruster et al., 2014). In tobacco (Nicoti-ana tabacum), the simultaneous overexpression of PsbS,violaxanthin de-epoxidase and zeaxanthin epoxidaseincreased the rate ofNPQ relaxation,which subsequentlyincreased growth in the field by 14% to 20% (Kromdijket al., 2016). These results prove that slowNPQ relaxationis an important limitation in naturally fluctuating irra-diance. Further, the results of Kromdijk et al. (2016) are apowerful testament to the fact that irradiancefluctuationsstrongly diminish growth in the field; they provide aglimpse into growth accelerations that would be possibleif the rate constants of other processes responding tofluctuating irradiance were enhanced.

Chloroplast Movement

Another potential limitation to electron transportunder fluctuating irradiance is the movement of

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chloroplasts in response to blue irradiance. At high blueirradiance, chloroplasts move toward the anticlinal walls ofthemesophyll cells,while at lowblue irradiance, theymoveto the periclinal walls (Haupt and Scheuerlein, 1990),resulting in decreases and increases of absorptance, re-spectively (Gorton et al., 2003; Williams et al., 2003; Tholenet al., 2008; Loreto et al., 2009). In leaves of some species,chloroplast movements can change irradiance absorptanceby.10%, although in other species the effect is,1% (Daviset al., 2011). The reduction in absorptance in high irradiancehas a photoprotective effect, and significant reductions inphotoinhibition have been demonstrated for Arabidopsis(Kasahara et al., 2002; Davis and Hangarter, 2012). Fur-thermore, chloroplast movements alter the area of chloro-plasts exposed to the intercellular spaces, changingmesophyll conductance (gm). Importantly, chloroplastsmove within minutes (Brugnoli and Björkman, 1992; Duttaet al., 2015; Łabuz et al., 2015), so the effects of theirmovement on absorptance and gm (Box 3) should be rele-vant under naturally fluctuating irradiance. In particular,slow chloroplast movement toward the low irradianceposition (time constants of 6–12min; Davis and Hangarter,2012; Łabuz et al., 2015), which lead to increased absorp-tance, would transiently decrease absorptance after dropsin irradiance, thus limiting electron transport and photo-synthesis (i.e. similar to the effect of slow qE relaxation, seeabove). However, experimental evidence of this possiblelimitation is currently lacking.

Enzyme Activation and Metabolite Turnover

The activity of several key enzymes in the CalvinBenson cycle (CBC) is regulated in an irradiance-

dependent manner, much of which depends on thethioredoxin (TRX) system (Geigenberger et al., 2017).There is a multitude of TRX types and isoforms. Forexample, Arabidopsis chloroplasts contain 10 differentTRX isoforms (Michelet et al., 2013). Chloroplastic TRXsmay be reduced by ferredoxin-dependent or NADPH-dependent thioredoxin reductases (Nikkanen et al.,2016; Thormählen et al., 2017). In the chloroplast, f-typeTRXs control the activation state of Fru-1,6-bisphos-phatase (FBPase), sedoheptulose-1,7-bisphosphatase(SBPase), and Rubisco activase (Rca; Michelet et al.,2013; Naranjo et al., 2016). While oxidized FBPasemaintains a basal activity of 20% to 30%, the oxidizedform of SBPase is completely inactive (Michelet et al.,2013). In Pisum sativum, the activities of phosphoribu-lokinase (PRK) and glyceraldehyde-3-phosphate de-hydrogenase are controlled by the redox-regulatedprotein CP12, which binds the enzymes together in lowirradiance and thereby inactivates them even if they arereduced (i.e. active; Howard et al., 2008). However, thistype of regulation by CP12 is not universal, as in severalspecies, the complex formed by CP12, glyceraldehyde-3-phosphate dehydrogenase, and PRK was mostly ab-sent in darkness or the enzymes existed both in thebound and free form (Howard et al., 2011). Apart fromthe action of CP12, PRK activity is also regulated byTRX m and f (Schürmann and Buchanan, 2008).

Within the first minute after a switch from low tohigh irradiance, SBPase, FBPase, and PRK are believedto limit photosynthesis via the slow regeneration of ri-bulose-1,5-bisphosphate (RuBP; Sassenrath-Cole andPearcy, 1992, 1994; Sassenrath-Cole et al., 1994; Pearcyet al., 1996). These enzymes activate and deactivatequickly, with time constants (t) of ;1 to 3 min for

Figure 2. Schematic depiction of dynamic reactions of leaf photosynthetic processes to irradiance fluctuations. The leaf is initiallyadapted to shade (50 mmol m22 s21), then exposed to strong irradiance (1,000 mmol m22 s21) for 60 min, after which it is shadedagain for 35 min. Displayed are net photosynthesis rate (A; red line, continuous), stomatal conductance (gs; blue line, dots),substomatal CO2 partial pressure (Ci; green line, long dashes), nonphotochemical quenching (NPQ; gray line, short dashes), andthe electron transport efficiency of PSII (FPSII; black line, long dashes and dots). These values are representative of Arabidopsis Col-0, grown in climate chambers at a constant irradiance of 170 mmol m22 s21.

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activation and ;2 to 4 min for deactivation(Supplemental Table S1). Compared to limitation byeither Rubisco or gs (see below), which often (co)-limitphotosynthetic induction for 10 to 60min, the limitationdue to activation of SBPase, FBPase, and PRK appearsnegligible but is relatively understudied. Due to theirrelatively quick deactivation in low irradiance, it maybe that in the field the activation states of these enzymesare a stronger limitation of CO2 assimilation thanRubisco or gs (Pearcy et al., 1996), as the majority ofsunflecks in canopies are short and narrowly spaced(see above). More research into this potentially largelimitation is needed, e.g. by using plants with increasedconcentrations of CBC enzymes (e.g. Simkin et al.,2015), as well as “always-active” FBPase and PRK(Nikkanen et al., 2016).The dependence of the activation state of Rubisco

upon irradiance resembles that of an irradiance re-sponse curve of photosynthesis (Lan et al., 1992). In lowirradiance, 30% to 50% of the total pool of Rubisco isactive (Pearcy, 1988; Lan et al., 1992; Carmo-Silva andSalvucci, 2013). The remainder is activated with a t of3 to 5 min after switching to high irradiance (Pearcy,1988; Woodrow and Mott, 1989; Kaiser et al., 2016;Taylor and Long, 2017). Activation of Rubisco active

sites requires the binding of Mg2+ and CO2 to form acatalytically competent (carbamylated) enzyme, afterwhich RuBP and another CO2 or O2 molecule have tobind for either carboxylation or oxygenation to occur(Tcherkez, 2013). Rubisco activates more quickly athigher CO2 partial pressures, both in folio (Kaiser et al.,2017) and in vitro (Woodrow et al., 1996), a phenome-non that is not well understood and whose kineticscannot be explained by carbamylation.

Several types of sugar phosphates can bind toRubisco catalytic sites and block their complete acti-vation (Bracher et al., 2017). Removal of these inhibitorsrequires the action of Rca (Salvucci et al., 1985), whoseactivity depends on thioredoxin and ATP. Rca light-activates with a t of ;4 min in spinach (Spinacia oler-acea; Lan et al., 1992). In Arabidopsis, Rca is present intwo isoforms, of which the larger, a-isoform is redoxregulated and the smaller, b-isoform is regulated by thea-isoform (Zhang and Portis, 1999; Zhang et al., 2002). Intransgenic plants only containing the b-isoform, photo-synthetic induction after a transition from low to highirradiancewas faster than in thewild type, asRca activitywas constitutively high and independent of irradiance(Carmo-Silva and Salvucci, 2013; Kaiser et al., 2016).Modifying the composition of Rca (Prins et al., 2016) or

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its concentration, either transgenically (Yamori et al.,2012) or through classical breeding (Martínez-Barajaset al., 1997), might enhance photosynthesis and growthin fluctuating irradiance (Carmo-Silva et al., 2015).

After the fixation of CO2 into RuBP, the triose phos-phates may be transported out of the chloroplast andconverted into sugars, after which the phosphate istransported back into the chloroplast and recycled viathe chloroplast ATPase and the CBC (Stitt et al., 2010).The enzyme Suc phosphate synthase can transientlylimit photosynthesis after a transition from low to highirradiance, but this has so far only been shown in ele-vated CO2 (Stitt and Grosse, 1988). After decreases inirradiance, pools of CBC intermediates can transientlyenhance photosynthesis (“postillumination CO2 fixa-tion”), while the turnover of Gly in the photorespiratorypathway may be visible as a transient decrease inphotosynthesis (“postillumination CO2 burst”). Aftervery short (#1 s) sunflecks, postillumination CO2 fixa-tion enhances total sunfleck carbon gain greatly, suchthat the CO2 fixed directly after a sunfleck exceeds theCO2 fixed during the sunfleck (Pons and Pearcy,1992). The negative effect of postillumination CO2fixation on the carbon balance of a sunfleck seems lesspronounced in comparison (Leakey et al., 2002). Formore details on both phenomena, see Kaiser et al.(2015).

CO2 Diffusion into the Chloroplast

Diffusion of CO2 to the site of carboxylation is me-diated by gs and gm. Stomata tend to decrease theiraperture in low irradiance, when evaporative demandand demand for CO2 diffusion are small. Vast differ-ences exist between species (15- to 25-fold) for steady-state gs in low and high irradiance (e.g. McAuslandet al., 2016), for rates of stomatal opening after irradi-ance increases (t = 4–29 min) and for rates of stomatalclosure after irradiance decreases (t = 6–18 min; Vicoet al., 2011). Often, initial gs after a switch from low tohigh irradiance is small enough, and stomatal openingis slow enough (Fig. 2), to transiently limit photosyn-thesis (McAusland et al., 2016; Wachendorf and Küp-pers, 2017). Manipulating gs to respondmore quickly toirradiance could greatly enhance photosynthesis andWUE in fluctuating irradiance (Lawson and Blatt, 2014;Vialet-Chabrand et al., 2017b). Mesophyll conductancewill further affect the CO2 available for photosynthesis(Tholen et al., 2012; Yin and Struik, 2017), and steady-state gm affects CO2 diffusion as strongly as does gs(Flexas et al., 2008, 2012). Mesophyll conductance maybe variable under fluctuating irradiance (Campanyet al., 2016), as some of the processes determining gm areflexible (Price et al., 1994; Flexas et al., 2006; Uehleinet al., 2008; Otto et al., 2010; Kaldenhoff, 2012). Thepossibility that transient gm changes limit photosyn-thesis in fluctuating irradiance is discussed in Box 3.

Limiting CO2 diffusion into the chloroplast after aswitch from low to high irradiance may transiently

limit photosynthesis in two ways: via a transiently lowavailability of the substrate CO2 for carboxylation andby decreasing the rate of Rubisco activation (Mott andWoodrow, 1993). While the former limitation is visiblethrough a concomitant increase in A and chloroplastCO2 partial pressure (Cc) along the steady-state A/Ccrelationship (Küppers and Schneider, 1993), the lattercan be calculated by log-linearizing CO2 assimilationafter an increase in irradiance, after correcting forchanges in Ci (Woodrow andMott, 1989). The apparentt for Rubisco activation calculated from gas exchange infolio correlates well with Rubisco activation in vitro(Woodrow and Mott, 1989; Hammond et al., 1998) andwith Rca concentrations (Mott and Woodrow, 2000;Yamori et al., 2012). Additionally, Rubisco activationduring photosynthetic induction can be approximatedby “dynamic A/Ci curves” which are achieved bymeasuring the rate of photosynthetic induction at sev-eral Ci levels and plotting maximum rates of carboxyl-ation (Vcmax) as a function of time (Soleh et al., 2016). Itwas recently shown that the apparent tRubisco derivedfrom dynamic A/Ci curves was in agreement withvalues derived using the procedure described byWoodrow andMott (1989) and Taylor and Long (2017).Apparent tRubisco decreases with increases in Ci (Mottand Woodrow, 1993; Woodrow et al., 1996) and withrelative air humidity (Kaiser et al., 2017) during pho-tosynthetic induction. The latter phenomenon wascaused by humidity effects in initial gs, leading to fasterdepletion of Cc and transiently lower Cc after an in-crease in irradiance (Kaiser et al., 2017). The mechanismbehind this slowing down of Rubisco activation due tolower Cc is as yet unresolved.

PHENOTYPING FOR FASTER PHOTOSYNTHESIS INFLUCTUATING IRRADIANCE

High-throughput phenotyping for natural variation(including mutant screens, e.g. Cruz et al., 2016) gainedimportance following the analyses of Lawson et al.(2012), Lawson and Blatt (2014), and Long et al. (2006).These studies highlighted the response times of pho-tosynthesis to changing irradiance as limitations tocarbon gain, including the slow response of gs (Tinoco-Ojanguren and Pearcy, 1993), which can also diminishWUE (Lawson and Blatt, 2014), stressing their value asroutes for improving assimilation. Kromdijk et al.(2016) consequently showed that improved relaxationof qE type NPQ improved tobacco yield under fieldconditions. While they used transgenics, the modifica-tions used—increased amounts of PsbS, violaxanthinde-epoxidase and zeaxanthin epoxidase—could haveoccurred naturally. In fact, altering gene expressionpatterns has been a major route to improving the use-fulness of plants for agriculture (Swinnen et al., 2016),either through natural variation in the gene pool ofnatural ancestors or through mutations occurring dur-ing domestication. Naturally occurring variation in atrait can be used to analyze the genetic architecture of

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the trait, and this can be used to increase the efficiencyof improving the trait by breeding. Knowing how a traitis genetically determined increases the options for itsimprovements by breeding beyond those emergingfrom the physiological or biochemical approaches ofthe kind used by Kromdijk et al. (2016). Variation forthe kinetics of photosynthetic responses to changingirradiance is also another resource for further conven-tional physiological and biochemical analyses of theregulation and limitations acting on photosynthesisunder these conditions.If variation for a quantitative trait, such as photo-

synthetic responses, is identified in a genetically diversepopulation, and the genetic diversity has been mappedby means of e.g. single nucleotide polymorphisms, it ispossible to correlate genetic with phenotypic variation(e.g. Harbinson et al., 2012; Rungrat et al., 2016) and toidentify the QTL (quantitative trait loci) whose varia-tion correlates with phenotypic variation. Differenttypes of mapping populations can be used for QTLidentification: genome-wide association study andlinkage mapping using recombinant inbred lines (RIL).These strategies have their own advantages and dis-advantages (Bergelson and Roux, 2010; Harbinsonet al., 2012; Korte and Farlow, 2013; Rungrat et al.,2016). Once identified, QTL are invaluable as markersfor conventional plant breeding approaches and as astarting point for identifying the causal gene for theQTL. It is obviously advantageous to maximize thechances of finding an association by including as muchgenetic diversity as possible in a mapping population.In the case of crop plants, domestication results in a lossof genetic diversity (Doebley et al., 2006; Shi and Lai,2015), so there is much to be gained by including fieldraces and wild types in the construction of mappingpopulations or RILs. The phenotypic data required forQTL mapping requires measurements upon hundredsor thousands of individuals depending on the mappingapproach adopted, the precision of the phenotypingprocedure compared to the variability of the trait, andthe heritability of the trait. In photosynthesis, whicheven in stable environments can change diurnally,quick measurements are needed (Flood et al., 2016).Measuring this many plants quickly places consider-able demands on the design of high-throughput sys-tems. Currently, the measuring technologies that arebest suited to automated high-throughput phenotypingof plant photosynthetic traits, including those inunstable irradiance, are chlorophyll fluorescenceimaging (Barbagallo et al., 2003; Furbank and Tester,2011; Harbinson et al., 2012; Rungrat et al., 2016) andthermal imaging for measuring stomatal responses(Jones, 1999; Furbank and Tester, 2011; McAuslandet al., 2013). While it is based on fluorescence from PSII,chlorophyll fluorescence allows the measurement ofmany useful photosynthetic parameters such as theelectron transport efficiency of PSII, NPQ, and itscomponents (of which qE is most commonly reported),Fv/Fm, qP, Fv9/Fm9, and similar parameters (Baker et al.,2007; Furbank and Tester, 2011; Harbinson et al., 2012;

Murchie and Harbinson, 2014). Chlorophyll fluores-cence procedures are well developed, and the phenom-enology and correlations of fluorescence-derivedphysiological parameters are well understood (e.g. Bakeret al., 2007; Baker, 2008; Murchie and Harbinson, 2014).Biomass accumulation can also be used as a measure ofplant fitness, and while this is not high-throughput norspecific for a photosynthetic process, it is simple to apply,requires no specific technology, and gives a useful mea-sure of the extent to which a plant can successfully adaptto fluctuating irradiance.

While the technologies and procedures for pheno-typing and QTL identification are promising, theapplication of this approach to photosynthesis is stilllimited, especially in the case of photosynthetic re-sponses to fluctuating irradiance. QTL for qE havebeen identified using low throughput phenotyping(Jung andNiyogi, 2009). van Rooijen et al. (2017) haveidentified a gene (YS1) underlying longer-term re-sponses to an irradiance change using a genome-wideassociation study analysis of an Arabidopsis map-ping population (Li et al., 2010). This work demon-strates that phenotyping combined with furthergenetic analysis can be used for identifying QTLs andgenes linked to variation in a photosynthetic trait,opening the door to a new approach to understandingphotosynthetic responses to fluctuating irradiance. If aQTL can be found for a trait, such as faster responses tofluctuating light, then by implication there is an associa-tion with genetic markers. This association can be used inmarker-assisted breeding to accelerate the transfer of the

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QTL into a genotype that lacks the trait but has otherwisedesirable properties.

CONCLUSION

Average rates of photosynthesis decrease underfluctuating irradiance when compared to a constantenvironment.Whereas part of this decrease is explainedby the nonlinear response of photosynthesis to irradi-ance, further decreases are the result of slow changes inenzyme activities, stomatal conductance, and NPQ.Changes in mesophyll conductance and irradiance ab-sorbance (caused by chloroplast movements) may addto these limitations, but this awaits experimental veri-fication. Whereas much of the earlier research focusedon Rubisco activity and dynamic stomatal conductance,recent experimental and modeling studies suggestother processes (and enzymes) to be limiting (Hou et al.,2015; Guo et al., 2016). Therefore, both models and ex-periments should widen their scope. This requiresextending the toolbox of the dynamic photosynthesisexperimentalist to include rapid gas exchange systems,chlorophyll fluorescence, and spectroscopic techniquesand the design of new measurement protocols andmathematical models to provide the necessary param-eters. There is also the realization that the growth en-vironment of plants should approximate thatexperienced in the field (Poorter et al., 2016). Recentdevelopments of lighting technology (LEDs) enablethis. Increasingly, plants are grown under more fluc-tuating conditions (Külheim et al., 2002; Leakey et al.,2003; Athanasiou et al., 2010; Alter et al., 2012; Vialet-Chabrand et al., 2017a), but the complex nature ofnatural irradiance fluctuations and the scarcity ofmeasurements in the field mean that to date no stan-dard exists for defining relevant fluctuating growthconditions in the laboratory.

Our review of the literature indicates that the fluc-tuating regime strongly depends on whether fluctua-tions are caused by wind and gaps in the canopy (i.e.sunflecks) or by intermittent cloudiness (i.e. cloud-flecks; Box 2). Whereas the former consists of fluctua-tions at the scale of seconds over a low irradiancebackground, cloudflecks are fluctuations at the scale ofminutes over a high irradiance background. Further-more, the variation across species, canopy structure,and location seems to be small, but further characteri-zation of cloudflecks and sunflecks is needed. Bothfluctuating regimes are relevant to crops in the field, butthe relative importance of processes limiting photosyn-thesis could depend on the specific irradiance pattern.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. Time constants of irradiance-dependent activa-tion and deactivation of FBPase, PRK, and SBPase, based on fits topublished data.

Received September 6, 2017; accepted October 16, 2017; published October 18,2017.

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Update on Dynamic Photosynthesis in Crops

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