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Article Physiological Characteristics of in -, Green and Dark-Green Chinese Kale (Brassica oleracea L. var. alboglabra Musil.) under Varying Intensities

Kuan-Hung Lin 1 , Feng-Chi Shih 2, Meng-Yuan Huang 2,* and Jen-Hsien Weng 2,*

1 Department of and Biotechnology, Chinese Culture University, Taipei 111, Taiwan; [email protected] 2 Department of Life Sciences, National Chung-Hsing University, Taichung40227, Taiwan; [email protected] * Correspondence: [email protected] (M.-Y.H.); [email protected] (J.-H.W.)

 Received: 8 July 2020; Accepted: 24 July 2020; Published: 30 July 2020 

Abstract: The objective of this work was to study physiological characteristics and photosynthetic apparatus in differentially pigmented of three Chinese kale . Chlorophyll (Chl) fluorescence and photochemical reflectance index (PRI) measurements in green, yellow-green, and dark-green cultivars in response to varying light intensities. As light intensity increased from 200 to 2000 photosynthetic photon flux density (PPFD), fraction of light absorbed in (PS) II and PRI values in all plants were strongly lowered, but fraction of light absorbed in PSII dissipated via thermal dissipation and non-photochemical quenching (NPQ) values in all plants wereremarkably elevated.When plants were exposed to 200 PPFD, the values of fraction of light absorbed in PSII, utilized in photosynthetic electron transport(p), andfraction of light absorbed excitation energy in PSII dissipated via thermal energy dissipation (D), remained stable regardless of the changes in levels of Chla + b. Under 800 and 1200 PPFD, the values of p and electron transport rate (ETR) decreased, but D and NPQ increased as Chla + bcontent decreased, suggesting that decrease inChla + bcontent led to lower PSII efficiency and it became necessary to increase dissipate excess energy. On the contrary, in 2000 PPFD, leaves with lower Chla + bcontent had relatively higher p and electron transport rate (ETR) values and lower D level, as well as tended to increase more in NPQ but decrease more in PRI values. The consistent relations between PRI and NPQ suggest that NPQ is mainly consisted ofthe cycle-dependentenergy quenching.Yellow-green showed lower Chla + bcontent but high /Chla + b ratio and had high light protection ability under high PPFD. The precise management of photosynthetic parameters in response to light intensity can maximize the growth and development of Chinese kale plants.

Keywords: chlorophyll fluorescence; energy dissipation; light response; ; photosynthesis efficiency; photosynthetic photon flux density; pigment

1. Introduction Photosynthesis is biochemically regulated to maintain the balance between the rates of its component processes and concentrations of metabolites in response to environmental changes. Eco-physiological studies require knowledge of the photosynthetic rates of plants under different environmental conditions and a broad range in light intensity. Plants respond to sudden and sustained fluctuations in light intensities, partly throughtheir molecular -signaling transduction mechanisms in the [1]. Light intensity not only affects growth and biochemical characteristics,

Plants 2020, 9, 960; doi:10.3390/plants9080960 www.mdpi.com/journal/plants Plants 2020, 9, 960 2 of 13 but also associates with the photosynthetic efficiency of plants. The study of photosynthesis irradiance relationships is a basic aspect of plant physiological research and is important for managing various species; photosynthetic light responses can be used to assess the ability to capture light and understand the optimal habitat light intensity conditions of plants [2]. Both chlorophyll fluorescence (ChlF) and reflectance spectroscopy (i.e., photochemical reflectance index, PRI) are noninvasive techniques andoften used in physiologicalstudies to investigate a plant0s response to various abiotic and biotic stresses in controlled environments and in the field [3]. The measurements of ChlF and PRI are simple and reliable methods for estimating the photosynthetic rate [4,5]. However, previous research also found that ChlF parameters were affected by the amount of photosynthetic pigments [6]. The effect of pigments on ChlF and PRI in Chinese kale species with different colors subjected todifferent light intensities has not yet been examined. Chinese kale (Brassica oleracea L. var. alboglabra) is a crucifer vegetable crop grown in Southeast Asian countries [7]. There are many cultivars with varied leaf colors, includingyellow-green, green, and dark-green. Most of the plant materials with low Chl contentscan be collected from nitrogen deficiency or restricted treatments, wheresome materials, such as Chl-deficientspecies or mutants, can be used for photosynthesis measurements under normal nutrition or full fertilization conditions.Therefore, it is worth to study the effect of pigments on ChlF and PRI in varied leaf colors of Chinese kale species under full and restrictedfertilization conditions at variousphotosynthetic photon flux densities (PPFD). However, there is limited information available regarding ChlF and spectral reflectanceof these plants under various light intensities. Understanding the photosynthetic characteristics of the Chinese kale would benefit field cultivation management and inform the relationship between leaf color and light energy distribution and utilization. The hypothesis of this study was that ChlFparameters might exhibit distinguishable differences among yellow-green, green, and dark-green Chinese kale plants under different light intensities since the pigment composition of their leaves differs. ChlF and PRI indices would be changed in varied leaf colors with different Chl contents in response to different PPFDs.The aim of this study was to determine the actual state of the photosynthetic apparatus in differently pigmented leaves of three Chinese kale cultivars. The appearance of the capture, transfer, and dissipation of excitation energy were detected by ChlF measurements and PRI values in green, yellow-green, and dark-green cultivars in response to varying light intensities. ChlF and PRI can be considered selection indexes for examining the growth of Chinese kaleplants at specific and optimal light intensities under artificial light illuminations. The relationship of ChlF and PRI indices with Chla + band carotenoids (Car) can be used for eco-physiological research in Chinese kaleplants. The precise management of ChlF and PRI parameters in response to light irradiances may hold promise for maximizing the economic efficiency of the growth, development, and pigment potential of Chinese kaleplants grown in controlled environments.

2. Results

2.1. Influence of Light Intensity on Chl a + band the Parameters of Chinese Kale Plants Figures1 and2 show thatphotosynthetic electron transport ( p) and PRI levels of all plantsdecreased, but thermal energy dissipation (D) and non-photochemical quenching (NPQ) values increased as light 2 1 intensity increased from 200 to 2000 µmol m− s− PPFD. In addition, relationships among p, D, excess energy (E), and PRI vs. Chla + b in all plants were varied with PPFDs. The p, D, and E values in all 2 1 plants exposed to 200 µmol m− s− were approximately 0.6, 0.4, and 0.1, respectively, and displayed no variation with the change of Chla + b (Figure1A,H,O). There were positive and significant correlations at r = 0.357, 0.555, and 0.510 between p and Chla + b in all tested plants in response to 400, 800, and 2 1 2 1 1200 µmol m− s− , respectively (Figure1B–D). The value of p in all plants under 2000 µmol m− s− for all irradiation times was below 0.2 (Figure1E–G) and displayed no relationship between p and Chl a + 2 1 b except for a negative correlation (r = 0.477, p < 0.05) between p and Chl a + b under 2000 µmol m− s− for 2 h (Figure1G). No correlations were observed between D and Chl a + b in all plants under 200, Plants 2020, 9, 960 3 of 13

2 1 400, and 2000 µmol m− s− for 20 min (Figure1I,L). Nevertheless, D was significantly and negatively 2 1 correlated with Chla + b at PPFDs of 800 and 1200 µmol m− s− (r = 0.464 and 0.336, respectively; Figure1J,K). The values of D werenon-linearly significant and positively correlated with Chla + b at 2 1 r = 0.336 and 0.554 under 2000 µmol m− s− for 1 h and 2 h, respectively (Figure1M,N). All E values were weakly but significantly and negatively correlated to Chl a + b (r = 0.484–0.346, Figure1P–R,T,U) 2 1 in response to all PPFDs, except for no relationships being observed under 200 and 2000 µmol m− s− for 20 min (Figure1O,S), indicating that a lower Chl a + b content resulted in an increased E and produced excess energy in PSII in response to different PPFDs. The trend of D against Chla + b in all 2 1 plants was opposite to p values in response to medium PPFDs (800 and 1200 µmol m− s− ) and high 2 1 PPFD (2000 µmol m− s− ), where medium and high PPFDs yielded relatively higher D values from 0.6 to 0.8 readings (Figure1J–N) compared to p values (0.1–0.4, Figure1C–G). These results demonstrate that leaves with lower Chl content showed lower p and led to increasing D to dissipated more excess light energy in PSII under medium PPFD condition. On the contrary, in high PPFD condition, leaves with lower Chla + bcontent had relatively higher p value and lower D level. The E values were less affected by Chl a + b under all tested PPFDs due to relatively low values. Correlations between Chl a + b and PRI measurements of all plant leaves with all treatments in response to PPFD conditions exhibited significant and positive r values (0.598–0.738, p < 0.001, Figure2A–G).In general, Chl a + b and PRI values in dark-green Chinese kale were relatively higher than in green and yellow cultivars in response to all PPFDs. As PPFD increased from 200 to 2000 µmol m 2s 1, PRI values decreased from 0.02 to 0.05 in response to lower leaf Chl a + b content − − − − (<0.1 gm 2), butincreased from 0.005 to 0.02 in higher leaf Chl a + b content (;0.4 gm 2). Thus, the − − − − slope of the linear equation in PRI vs. Chl a + b increased from 0.048 to 0.095 as light intensity increased 2 1 from 200 to 2000 µmol m− s− PPFD. NPQ vs. Chl a + b variations in response to light intensities inall plants are demonstrated in Figure2H–N. Data from all tested cultivars with all fertilization treatments show that the relationship between NPQ and Chl a + b was significantly and negatively correlated (r = 0.470–0.662) in all plants under medium and high PPFD conditions (Figure2K–N). Nevertheless, weakly positive (r = 0.346, p < 0.05) and no insignificant correlations were detected between NPQ 2 1 and Chla + b at 200 and 400 µmol m− s− , respectively (Figure3H,I). The trend of NPQ vs. Chl was 2 1 opposite to PRI vs. Chl. As PPFD increased from 200 to 2000 µmol m− s− ,NPQ values increased from 2 1 to 5 in response to lower leaf Chl a + b content (<0.1 gm− ), but NPQ values only increased from 1 to 2 2 in higher leaf Chl a + b content (;0.4 gm− ), suggesting that leaves of lower Chl content resulted in 2 1 increasing NPQ and dissipating more excess light energy under medium (800 and 1200 µmol m− s− ) 2 1 and high (2000 µmol m− s− ) PPFD conditions. No effects of fertilization (FF and RF) on Chl a + b vs. ChlF and PRI in all plants at all PPFDs were detected in this study. Plants 2020, 9, 960 4 of 13

Plants 2020, 9, x FOR PEER REVIEW 4 of 15

Figure 1.Relationships among Chla + b content, fraction of light energy absorbed in photosystem II Figure 1.thatRelationships is utilized in photochemistry among Chla (p+)(panelsb content, A–G), dissipated fraction ofthermally lightenergy (D)(panels absorbed H–N), and in excess photosystem II that is utilizedenergy ( inE)( photochemistrypanel O–U) in yellow (p-)green (panels (triangle, A–G), ▲ dissipatedand △), green thermally (circle, ● and (D ○)), (panels and dark H–N),-green and excess energy (E(square,) (panel ■ and O–U) □) foliage in yellow-green cultivars of Chinese (triangle, kale withN andrespect ),to greendifferent (circle, light intensityand treatments), and dark-green (PPFD of 200, 400, 800, and 1200μmol m−2s−1for 20 min, and 20004 μmol m−2 s−1conditions• for 20 min, 1 (square,  and ) foliage cultivars of Chinese kale with respect to different light intensity# treatments h, and 2 h). Black and white represents a plant treated with full fertilization (100 mL of 0.1%NH4NO3 2 1 2 1 (PPFD ofand 200, K2 400,HPO4 800, applied and twice 1200 weeklyµmol) and m− restricteds− for 20fertilization min, and (50 2000 mL ofµ 0.1mol% NH m−4NOs−3 andconditions K2HPO4 for 20 min, 1 h, and 2applied h). Black once andweekly white), respectively. represents Each a symbol planttreated represents with the average full fertilization of one leaf on (100 one mL plant of and 0.1%NH 4NO3 36 plants were randomly selected from each treatment. Each Chl index was calculated using and K2HPO4 applied twice weekly) and restricted fertilization (50 mL of 0.1% NH4NO3 and K2HPO4 yellow-green (triangle, ▲ and △), green (circle, ● and ○), and dark-green (square, ■ and □) with leaf applied once weekly), respectively. Each symbol represents the average of one leaf on one plant data (n= 36) from the model′s validation datasets. The correlation coefficient (r) and significance of and 36 plantsthe regressionwere randomlyare shown (* p selected< 0.05; ** p from< 0.01; *** each p< 0.001; treatment. ns, not significant Each Chl). index was calculated using yellow-green (triangle, N and ), green (circle, and ), and dark-green (square,  and ) with leaf 4 • data (n = 36) from the model0s validation datasets. The# correlation coefficient (r) and significance of the regression are shown (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant). Plants 2020Plants, 9, 960 2020, 9, x FOR PEER REVIEW 6 of 15 5 of 13

Figure 2.FigureRelationships 2.Relationships among among Chla Chla+ + bb content, photochemical photochemical reflectance reflectance index (PRI index)(panels (PRI) A–G (panels), A–G), and non-photochemical quenching (NPQ)(panels H–N) in yellow-green (triangle, ▲ and △), green and non-photochemical quenching (NPQ) (panels H–N) in yellow-green (triangle, N and ), green (circle, ● and ○),and dark-green (square, ■ and □) foliage cultivars of Chinese kale with respect to 4 (circle, differentand ),and light intensity dark-green treatments (square, (PPFD  of and 200, 400,) foliage 800, and cultivars1200μmol m of−2 Chineses−1for 20 min, kale and with respect • −2 −1 2 1 to different2000μmol light# m intensity s conditions treatments for 20 min, (PPFD 1 h, and of2 h) 200,. Black 400, and 800,white andrepresents 1200 a µplantmol treated m− withs− for 20 min, full fertilization2 1(100 mL of 0.1% NH4NO3 and K2HPO4 applied twice weekly) and restricted and 2000 µmol m− s− conditions for 20 min, 1 h, and 2 h). Black and white represents a plant treated fertilization (50 mL of 0.1% NH4NO3 and K2HPO4 applied once weekly), respectively. Each symbol with full fertilization (100 mL of 0.1% NH NO and K HPO applied twice weekly) and restricted represents the average of one leaf on one 4 plant3 and 36 plants2 were4 randomly selected from each fertilizationtreatment. (50 mL Each of Chl 0.1% index NH was4NO calculated3 and Kusing2HPO yellow4 applied-green ( oncetriangle, weekly), ▲ and △ respectively.), green (circle, Each● symbol representsand the ○), and average dark-green of one(square, leaf ■ onand one□) with plant leaf data and (n 36= 36 plants) from the were model randomly′s validation selecteddatasets. from each treatment.The Each correlation Chl indexcoefficient was (r) calculated and significance using of the yellow-green regression are (triangle,shown (* p< N0.05;and ** p<), 0.01; green *** p (circle,< and 0.001; ns, not significant). 4 • ), and dark-green (square,  and ) with leaf data (n = 36) from the model0s validation datasets. The# correlation coefficient (r) and significance of the regression are shown (* p < 0.05; ** p < 0.01;

*** p < 0.001; ns, not significant). Plants 2020Plants, 9, x FOR2020 ,PEER9, 960 REVIEW 8 of 15 6 of 13 PlantsPlants 20 20,20 9Plants,, x9 ,FOR x FOR 20 PEER20 PEER, 9, xREVIEW FOR REVIEW PEER REVIEW 8 of8 of15 15 8 of 15

FigureFigureFigure 3. 3. 3.Figure TheFigure TheThe responses responses 3.responses 3.The The responses ofresponses of of electron electron electron ofelectron of transportrate transportrate transportrateelectron transportrate transportrate ( ETR(ETR(ETR) (ETR)to)to) to photosynthetic photosynthetic photosynthetic to(ETR photosynthetic)to photosynthetic photon photon photon photon flux flux flux photon flux density density density density flux (PPFD) density (PPFD(PPFD(PPFD)in)in) inin yellow ( yellowPPFD yellow yellow-green-green)-ingreen-green yellow ( ( ( )-,green) ,),) green, green green green ( ( ((), ) green,),) ,)and , and andand ( dark dark-green dark dark)-, green-andgreen-green dark ( (( -)greenfoliage) foliagefoliage)foliage ( cultivars cultivars cultivars cultivars)foliage of of cultivars of Chinese Chinese Chinese Chinese kaleof kale kale kale Chinese cultivated kale cultivatedcultivatedcultivated underunder under under ((A A( )A()A full)full)full fertilization fertilization fertilization treatment treatment treatment treatment ( (100 100( 100(100 mL mL mL mL of ofof of0.1 0.1%0.1 0.1%% %NH NHNH NH4NO4NONO4NO3 3and 3and and K KK 2KHPO2HPO2HPO4 4applied 4applied appliedapplied twice twice twicetwice weekly) cultivated under (A)full fertilization treatment (100 mL of4 0.1%3 NH4NO2 3 and4 K2HPO4 applied twice weeklyweeklyweekly) )and )and andand(B(B ()B )restricted )restricted restricted fertilization fertilization fertilizationfertilization treatment treatment treatment(50treatment(50(50(50 mL mL mLmL of of of0.1 0.1 0.1%0.1%% %NH NH NH4NO4NO4NO3 3and 3and andand K K2KHPO2HPO2HPOHPO4 4applied 4applied appliedapplied once once once weekly). weekly) and(B) restricted fertilization treatment(50 mL of4 0.1%3 NH4NO2 3 and4 K2HPO4 applied once µ −2−2−22 1 weeklyweeklyweekly).) .)Measurements .MeasurementsMeasurements Measurementsweekly). Measurements were werewere were made mademade made werein inin indifferent didifferent differentmadefferent inlight lightlight lightdifferent intensities intensities intensities light at intensities at at200, 200, 200, 400, 400, 400, at800, 800, 800,800,200, and and andand400, 1200 1200 12001200800, μmol μmol andμmolmol m1200 m m − μmol s− for m−2 −1−1−1 20 min, and 2000 µmol−2 m−2−2−1−12−1s 1 PPFD conditions for 20 min (a), 1 h (b), and 2 h (c). Vertical bars indicate sssforforfor 20 20 20 min, min, min,s−1 forand and and 20 2000 2000 min,2000 μmol μmol andμmol m2000 m m s −s μmolsPPFDPPFDPPFD− m conditions −2conditions conditions s−1PPFD forconditions for for 20 20 20 min min min (for a( )a(,a) 1,20) , 1 h1 hmin (h b( b)(,b) (,and) a,and )and, 1 2 h 2 h2 h ( (bh c() )c(,. c) and.Vertical) .Vertical Vertical 2 h ( barsc )bars .bars Vertical bars standard deviations (n = 6). indicateindicateindicate standard standard standardindicate deviation deviation standarddeviations s (deviations n( n=(n =6= )6 .6) .) . s (n= 6). 2.2. ETR Values of Three Chinese Kale Cultivars underVariousLight Intensity Conditions 2.2.2.2.2.2. ETR ETR ETR V V alues2.2.Valuesalues ETR of of ofT T hreeVThreealueshree Chinese Chinese Chineseof Three K K aleK aleChineseale C C ultivarsCultivarsultivars Kale under underC underultivarsVVariousVariousarious underLLightLightightVarious I ntensityI ntensityIntensityLight C C IonditionCntensityonditiononditions sCs ondition s ETRETRETR values values valuesETR were were valueswere analyzed analyzed analyzed were analyzed under under under various various undervarious variouslight light light intensities intensities intensities light intensities ( Figure( Figure(Figure (Figure 3 3) 3.) .)Fo .Fo For3 rfull).r full full For fertilization fertilization fertilization full fertilization ( panel( panel(panel (panel A), ETR values were analyzedµ under various2 1 light intensities (Figure 3). For full fertilization (panel AA)A,) ,)the ,the the ETR ETR theETR of ETRof of all all all ofplants plants plants all plants under under under under 2 200 20000μmolμmol 200μmolmmmolm−2−s2−−s21s−PPFD1− mPPFD1PPFD− s− did did didPPFD not not not show didshow show not any any any show differences. differences. differences. any diff erences.The The The highestETR highestETR highestETR The highest ETR A), the ETR of all plants2 under1 200μmolm−2s−1PPFD did not show any differences. The highestETR2 1 values at 118 µ−2−mol2−−21−1−1 em− s− were found in green and dark-green plants under−2− 8002−−21−1−1 µmol m− s− valuesvaluesvalues at at at118 values118 118 μmol μmol μmol at 118e e memm μmolsss were were were em found− 2found sfound−1 were in in in green foundgreen green and and inand greendark darkdark- greenand-green-green dark plants plants plants-green under8 under8 under8 plants000000μmol under8μmolμmolmmm00ssμmolsPPFDPPFDPPFDm, −,2 ,s −1PPFD, andandand both both bothPPFD, green green green and and and and both dark dark dark- greengreen-green-green and leaves leaves leaves dark-green had had had significantly significantly significantly leaves had higher higher higher significantly ETR ETR ETR values values values higher compared comparedcompared ETR valuestototo yellow yellow yellow comparedto and both green and dark-green leaves hadµ significantly2 1 higher ETR values comparedto yellow leavesleavesleaves under underyellow under 400, 400, 400, leaves 800, 800, 800, under and and and 1200 12001200 400,μmolμmolμmol 800,mmm− and2−s2−−s21s−PPFDs.1−PPFDs.1 1200PPFDs. However,mol However, However, m− s − relatively relatively relativelyPPFDs. higher higherHowever, higher ETR ETR ETR relatively values values values were were were higher ETR leaves under 400, 800, and 1200μmolm−2s−1PPFDs. However, relatively2 1 higher ETR values were values were found in yellow-green plants subjected−2−2−−21−1−1 to 2000 µmol m− s− PPFDs compared to green foundfoundfound in in in yellow yellow yellow-green-green-green plants plants plants subjected subjected subjected to to to 2000 2000 2000μmolμmolμmolmmmsssPPFDsPPFDsPPFDs− compared2 compared −compared1 t ot ot greeno green green and and and dark dark dark-green-green-green andfound dark-green in yellow plants.-green plants Furthermore, subjected ETR to 2000 valuesμmol ofm alls plantsPPFDs grown compared under to restricted green and fertilization dark-green plants.plants.plants. Furthermore, Furthermore, Furthermore,plants. Furthermore, ETR ETR ETR values values values ETR of of of all valuesall all plants plants plants of grown allgrown grown plants under under under grown restricted restricted restricted under fertilizationrestricted fertilization fertilization fertilization ( panel( panel(panel B B )B )were )(were panelwere near near nearB) were near tototo those those those (panel of of of full full full B) fertilization fertilization fertilization were near condition. to condition. condition. those of Dark Dark fullDark- fertilizationgreen-green-green plants plants plants condition. had hadhad the the the lowest Dark-greenlowestlowest ETR ETRETR value plants valuevalue ( had≒(≒(≒505050 theμmol μmolμmol lowest ETR to those of full fertilization2 1 condition. Dark2 1-green plants had the lowest ETR value (≒50 μmol −2−2−−21−1−1 value (;50 µ−2mol−2−−21−1−1 em− s− ) at 200 µmol m− s− PPFD and the value increased greatly−2−2−−21−1− to1 the peak eememmsss)at)at) at2 200200−00μmol2μmolμmol−1 mmmsssPPFDandPPFDandPPFDand−2 − 1 the the the value valuevalue inc inc increasedreasedreased greatly greatlygreatly to toto the thethe peak peak peak ( 120 (120(120 μmol μmol μmol e em emmsss) )at ) at at −2 −1 em µs )at 2002μmol1 m s PPFDandµ 2 the1 value increased greatly to the peak (120 µ μmol em2 s 1) at 112120020000μmolμmolμmol(120mmm−2−s2−−s21mols−PPFD,1−PPFD,1PPFD, em then− then thens− the) the atthe 1200value value value decreasedmol decreased decreased m− s− slow slow PPFD,slowlylyly to tothen to 80 80 80 μmol theμmol μmol value e em emm−2 decreased−s2−-s21s-at1-at1 at20 20 20000000μmol slowlyμmolμmolmmm− to2−s2−−s 8021s−PPFDfor1−PPFDfor1PPFDformol em − s− at 1200μmolm−2s−1PPFD,1 then the value decreased slowly to 80 μmol em−2s-1at 2000μmolm−2s−1PPFDfor 2000 µmol m− s− PPFD for 2 h. Both green and yellow-green plants showed no significant differences 2h.2h.2h. Both Both Both green2h. green green Both and and and green yellow yellow yellow and-green-green- green yellow plants plants plants-green showed showedshowed plants no noshowed no significant significant significant no significant differenc differenc differences es differences in inin ETR ETR ETR esunder under underin ETR all all all lightunder light light all light irradiances,irradiances,irradiances,in ETR except except except under that that all that light yellow yellow yellow irradiances,-green-green-green leaves leaves except leaves had that had had yellow-greensignificantly significantlysignificantly leaves higher higherhigher hadETRETRETR significantly value value value ( 110(110(110 higher μmol μmol μmol ETR value irradiances, except2 1 that yellow-green leaves had2 significantly1 higherETR value (110 μmol −2−2−−21−1−1 (110 µmol em− s− ) than other− plants2−2−−21−1−1 at 2000 µmol m− s− PPFD for 2 h. eememmsss)than)than)thanem other −other 2others−1) thanplants plants plants other at at at20 20plants 20000000μmolμmolμmol at m20mm00ssμmolsPPFDPPFDPPFDm for− for2 sfor− 12h. PPFD2h. 2h. for 2h.

Plants 2020, 9, x FOR PEER REVIEW 9 of 15

Plants2.3. 2020Relationships, 9, 960 betweenChl a + b and Car/Chl a + b 7 of 13 Relationship betweenChla + band Car/Chla + bin varied leaves is presented in Figure 4. 2.3.Regression Relationships analys betweenChlis showed a + thatb and Car Car/Chl/Chl w a as+ bsignificantly,strongly,and positivelycorrelated with Chla + b at r = 0.962.Moreover, yellow-green leaf had low Chl a + b content and high Car/Chla + Relationship betweenChla + band Car/Chla + bin varied leaves is presented in Figure4. Regression bratio. analysis showed that Car/Chl was significantly, strongly, and positivelycorrelated with Chla + b at r = 0.962. Moreover, yellow-green leaf had low Chl a + b content and high Car/Chla + bratio.

FigureFigure 4. 4.RelationshipsRelationships between between Chla Chl+a +b b content content and and Car Car/Chl/Chla +abin + b yellow-greenin yellow-green (triangle, (triangle,N and ▲ and), 4 green△), green (circle, (circle,and ● and), and ○) dark-green, and dark-green (square, (square, and ■ and) foliage □) foliage cultivars cultivars of Chinese of Chinese kale. Blackkale. andBlack • whiteand representswhite represents a plant# a beingtreated plant being withtreated full with fertilization full fertilizat (100 mLion of(100 0.1% mL NH of4 NO0.1%3 andNH K4NO2HPO3 and4 appliedK2HPO twice4 applied weekly) twice and weekly restricted) and fertilization restricted (50 fertilization mL of 0.1% (50 NH mL4NO of 30.1and% KNH2HPO4NO4 3applied and K2 onceHPO4 weekly),applied respectively. once weekly Each), respectively. symbol represents Each symbol the average represents of one the leaf average on one of plantone leaf and on 36 one plants plant were and randomly36 plants selected were randomlyfrom each treatment. selected from Each eachChl index treatment. was calculated Each Chl using index yellow-green was calculated (triangle, usingN andyellow),- greengreen (circle,(triangle,and ▲ and), △ and), green dark-green (circle, (square,● and ○), andand dark)- withgreen leaf (square, data (■n a=nd6) □ from) with the leaf 4 • modeldata 0(sn validation= 6) from the datasets. model′ Thes# validation correlation datasets. coefficient The (r)correlation and significance coefficient of the(r) and regression significance are shown of the (***regressionp < 0.001). are shown (* p< 0.05; ** p< 0.01; *** p< 0.001; ns, not significant).

3.3. Discussion Discussion RoutesRoutes for for light light energy energy absorbed absorbed by by Chl Chl molecules molecules in in photosynthetic photosynthetic tissue are are used used to to drive drive photosyntheticphotosynthetic processes, processes, dissipate dissipate heat, heat, and and re-emit re-emit light light energy energy such such as as ChlF ChlF [8 [8]]. Measuring. Measuring the the yieldyield of of ChlF ChlF gives gives specific specific information information about photochemicalabout photochemical efficiency efficiency and heat anddissipation. heat dissipation. Changes inC ChlFhanges can in be ChlF used can to quicklybe used assessto quickly plant assess physiological plant physiological responses during responses stress during [9]. Yang stress et[9] al.. Yang [10] studiedet al. [10] seasonalstudied relationships seasonal relationships between ChlFand between photosynthesis ChlFand photosynthesis at ecosystem scalesat ecosystem and showed scales how and leafshow ChlFed washow linked leaf ChlF with was -scale linked with solar-induced canopy‐scale ChlF solar in‐ ainduc temperateed ChlF in a temperate forest, indicatingdeciduous thatforest, ChlF indicat can being used that to ChlF track can photosynthetic be used to track rates photosynthetic at leaf, canopy, rates and ecosystemat leaf, canopy, scales. and Since ecosystem PSII is thoughtscales. Since to play PSII a key is thought role in the to responseplay a key of PSrole to in environmental the response stress,of PS to the environmental analysis of ChlF stress, under the varyinganalysis light of ChlF intensities under might varying reflect light PSII intensities behavior. might In this reflect study, PSII three behavior. Chinese kaleIn this cultivars study, with three variedChinese leaf kale colors cultivars were used with to varied elucidate leaf thecolors characteristics were used to of elucidate Chla + bas the related characteristics to ChlF under of Chl PPFDs.a + bas 2 1 relatedAs light to ChlF intensity under increased PPFDs. from 200 to 2000 µmol m− s− PPFD, lower p levels, as well as higher D andAs NPQ light values intensity were increased observed infrom all plants.200 to 2000 Plantsμmol can m absorb−2s−1PPFD, more lower photons p levels, under as high well irradiance as higher intensitiesD and NPQ than canvalues be used were in observed carbon fixation in all reactions plants. [ Plants11]. This can excess absorb absorbed more light photons energy under becomes high a stressor and enhances the formation of reactive species (ROS) that can damage many cellular

Plants 2020, 9, 960 8 of 13 components and therefore cause a depression in photosynthetic efficiency, especially in PSII [12]. In our study, the excess energy in PSII increased, leading to increases in D and NPQ values and decreases in p values due to greater energy dissipation when plants were exposed to higher PPFDs. The relationship between Chla + b and ChlF in all plants were varied with PPFDs.When plants were subjected to low 2 1 light intensities (200 µmol m− s− ), the values of p and D remained stable regardless of the changes in levels of Chl a + b, indicating that allocation of absorbed light energy in PSII was less affected by Chla + b content under low PPFD. However, when these plants were measured under moderate light 2 1 intensities (800 and 1200 µmol m− s− PPFD), p values increased but D and E values decreased as Chl a + b content increased, suggesting that thermal energy dissipation took place more in antennae and that when low Chl content led to lower PSII efficiency it became necessary to increase D in order to dissipate excess energy. Any environmental and physiological factors resulting in decreased photosynthesis rates at a constant PPFD should be expected to have the potential to lead to a greater excess of absorbed light. It was reported that the leaves with lower Chl content showed a lower photosynthetic rate [6]. Thus, under moderate light intensities, leaves with low Chl content had low p values due to high energy dissipation. ETR is the product of PSII efficiency and absorbed light anddescribes the relative rate of electron transport through PSII [13]. In C4 plants, is restricted, absorbedphotons may be mostly used to drive the CO2 fixation, and PSII efficiency is always parallel to variation in quantum yield of CO2 fixation inmany cases [14,15]. In contrast, a significant correlation was found between ETR and gross photosynthetic rate under conditionsin C3 plants, which lessen the interference of photorespiration and other alternative pathways for electrons, such asnear-constant temperature, CO2 2 1 and O2 concentrations [16,17]. In our study, when plants were measuredat 400–1200 µmol m− s− PPFD, plants with deeper leaf colortended to have higher ETR values for higher efficiency of photosynthesis 2 1 (Figure3). Nevertheless, when plants were measuredat 2000 µmol m− s− PPFD, yellow-green leaves had higher p and ETR values. These results indicate that dark-green cultivarfavored moderate PPFDs while yellow-green cultivar was adapted under high light intensities. ETR was calculated from p, but why could the consistent relations between p and ETR not be found under different PPFDs? A possible reason is that ETR value was calculated from both p value and absorbed light, but plant leaves with higherChla + bcontent had higher light absorbance rates (i.e., the leaves had a light absorbance rate between 0.76 and 0.88 in the study). Chinese kale contains phenolic components, antioxidants, and free scavenging properties, and has been the subject of chemical and biological studies [18,19].The final goal of the study was to develop a year-round production system for healthy, fresh, nutritional, and economically feasible Chinese kale plants produced in a controlled environment. An optimal strategy of light intensity regulation will help in designing growth chambers and greenhouse light environments to obtain maximum economic benefits for growing Chinese kale plants and also improving field cultural practices for growing these plants in hydroponics as pesticide-free vegetables. Therefore, these ChlFcomponents can be used as indices to characterize the physiology of Chinese kale plants in response to different PPFDs. Light intensity not only influences the accumulation of photosynthetic pigments, but also mediates photo-physiological parameters in plant leaves.PRIincorporates reflectance at this band and correlates withtotal content and activity of xanthophyll cycle pigmentsand with PSII photochemical efficiency [20]. To better understand the eco-physiology of Chinese kale plants, the relationships of ChlF parameters with Chl a + b were analyzed by determining PRI.When the light intensity increased 2 1 from 800 to 2000 µmol m− s− , leaves with lower Chl a + b tended to show an increase in NPQ but a decrease in PRI (Figure2). Increased NPQ values includexanthophyll cycle dependent energy quenching (photo-protection) and photoinhibitory quenching (damage of PSII) [21,22]. The consistent relationships were observed among PRI, Chl a + b, and NPQ at moderate and high PPFDs (Figure2), suggesting that NPQ is mainly consisted of a down-regulation of PSII efficiency, whichis associated with the xanthophyll cycledependentenergy quenching, but not to photoinhibition. Therefore, it is Plants 2020, 9, 960 9 of 13 speculated that yellow-green Chinese kale plant has high light protection abilityunder high PPFDs. Moreover, the trend of increasing NPQ values (Figure2) is di fferent from that of D values (Figure1) resulted from the formulated calculations. NPQ is calculated from the maximal levels of fluorescence before (Fm) and during (Fm0) illumination in response to down-regulation of PSII. However, D is calculated from Fm0 and Fv0 in response to the fraction of light absorbed in PS II utilized in photosynthetic electron transport under illumination. In order to characterize the photosynthetic capacity of plants, the light-response relations and consequent base points produce very important parameters. Assessing these parameters under light intensity variations provides important tools for understanding how to improve the photosynthetic productivity of plants. The analysis of ChlF and PRI combination cansearch for the photosynthetic responses of B. oleracea diversity that can help to explore the most suitable species composition and the structure of stands to reduce the environmental stress impacts and climate changes on these species [23]. In our study, the leaves with very lower Chl contents had higher p values and lower PRI values under 2 1 2000 µmol m− s− PPFD conditions for 20 min, 1 h, and 2 h, suggesting that the photoinhibition did not take place, possibly resulted from the higher values of Car/Chla + b. Light intensity canaffect the accumulation of pigments like carotenoids (Cars) and Chla + b by stimulating the enzymatic activity of kale plants subjected to light-induced stress [24]. High light intensities often generate excess heat that must be removed from photosynthetic systems to prevent pant damages. Antenna pigments, like Car, absorb light and transfer this energy to Chla + b, which initiates the sequence of photochemical events of photosynthesis [25]. Cars also channel energy away from Chla + b as a photoprotectant [26]. In our study, Cars were important for energy dissipation, and the different cultivars exhibited individual abilities and specificities of ChlF in response to PPFDs. As a result, different genotypes show different responses, and they can be used as a PPFD-plant model to investigate pigment composition under PPFD exposure. Cars as photo-sensitizers and play important roles as scavengers of ROS. Figures1–3 illustrate that the e ffects of varying light intensities on their ChlF components and PRI in regard to photosynthetic activity differed, and moderate PPFDs favored dark-green plants while yellow-green plants were adapted under high light intensities. The trend of ETR and p values are similar, suggesting that species can be grown under specific and optimal light intensity. The genotypic differences might be related to adaptation mechanisms induced by varying light intensities. Moreover, the impact of fertilizer treatments (FF and RF) on the physiological characteristics of photosynthesis was evaluated to quantify whether FF or RF levels affected ChlF and PRI, and results showed that fertilizer treatments did not influence the relationships both ChlF and PRI vs. Chla + b in any cultivar at all PPFD conditions. Differing responses in leaf pigments for optimizing plant growth and development in a controlled-irradiance setting depended on the cultivar of Chinese kale. Various light intensity culture systems may be used to satisfy commercial requirements for rapid, large-scale, and precise management of B. oleracea plant production. In addition, the average time required to measure ChlF and PRI isvery short. This means that many hundreds of individual plants can be screened per day, providing ample opportunity for the discovery of individuals that exhibit greater quality. Chinese kale is an open pollinated plant that easily outcrosses with other crops, resulting in uncertain quality. Simple evaluations of photosynthesis can be made and relationships betweenheat dissipation, photosynthetic efficiency, and fluorescence can also be estimated. This knowledge could also be used in a breeding program resulting in the development of new cultivars adapted to high light intensity locations. Currently, we are using ChlF and PRI values to select for photosynthetic capacity in plants for stress tolerance. Plants 2020, 9, 960 10 of 13

4. Materials and Methods

4.1. Plant Materials and Cultural Practice of Chinese kale (Brassica oleracea L. var. alboglabra Musil.) yellow-greenleaf (cv. Huang Chiehlan), greenleaf (cv. Lu Chiehlan), and dark-greenleaf (cv. HeiChiehlan) were purchased from Mingfeng (Fengyuan, Taiwan), Fangyuan (Yunlin, Taiwan), and Known-You (Kaohsiung, Taiwan) Co., respectively, for our experiments. The medium usedwas a commercial potting mix of sand, peat moss, and Perlite 1:1:1 (v/v/v) (Known-You Co., Taipei, Taiwan). Seeds were germinated andgrown on plastic plug trays for 8–10 days until were 3 to 5 cm in height. Seedlings were then transplanted into free-draining pots (20 cm diameter, 15 cm depth, one plant per pot) and grown in a greenhouse at National Chung-HsingUniversity, Taichung, Taiwan (24◦080N, 120◦400E), during October–January in a controlled greenhouse. Plants were evenly spaced to promote similar growth rates and sizes and received regular water and . Plants were grown for two weeks and 12 uniformly sized plants of each variety were selected and randomly separated into two fertilization groups for subsequent experiments. The full fertilization (FF) treatment comprised six plants of each variety treated with the full amount (100 mL) of a liquid fertilizer of 0.1% NH4NO3 and K2HPO4 twice weekly for two weeks. The restricted fertilization (RF) treatment consisted of a second batch of six plants given 0.1% NH4NO3 and K2HPO4 (50 mL) once weekly for two weeks.

4.2. Determination of ChlFVariables The potted plants were moved to a dark room overnight and the middle portions of fully expanded young leaves of a plant were used for measurements. Dark-adapted plants were exposed to light stepwise from low to high levels of photosynthetic photon flux density (PPFD; i.e., 200, 400, 800, 2 1 1200, and 2000 µmol m− s− ) from a slide projector with a tungsten halogen lamp for 20 min and 2 1 an extra 1 h and 2 h for 2000 µmol m− s− only. The ChlF parameters of dark-acclimated all night (before sunrise), light-exposed leaves, and dark-adapted for 20 min (after illumination) were measured at ambient temperature with a portable fluorometer (PAM 2000, Heinz Walz, Effeltrich, Germany). − Samples were first adjusted to dark conditions to assure that all reaction centers were in an open state under minimal non-photochemical dissipation of excitation energy [27]. The values of minimal ChlF (Fo) and maximal ChlF (Fm) were respectively determined from overnight dark-adapted samples using modulated irradiation via a weak light-emitting diode beam (measuring light) and saturating pulse.For leaves under each level of illumination, the efficiency of open PSII units during illumination (Fv /Fm ) was calculated as (Fm Fo )/Fm , and the actual PSII efficiency (∆F/Fm’) was calculated as 0 0 0− 0 0 (Fm Ft)/Fm . Fo , Fm and Ft are the minimal, maximal and steady-state levels of fluorescence during 0− 0 0 0 each level of illumination, respectively. The former was measured after far- illumination, middle was measured by applying a saturating flash, and the latter was determined at each PPFD level. From these data, several parameters can be computed based on modulated fluorescence kinetics [15,28]. The non-photochemical quenching (NPQ) coefficient is NPQ = (Fm Fm )/Fm , p = ∆F/Fm ,D = 1 − 0 0 0 (Fv /Fm ), and E = 1 p D. p is the fraction of light absorbed in PSII utilized in photosynthetic − 0 0 − − electron transport. D is the fraction of light-absorbed excitation energy in PSII that is dissipated via thermal energy dissipation; E is the fraction of excess energy in PSII. Electron transport rate (ETR) was calculated as ∆F/Fm PPFD 0.5 α [8]; α is the value of leaf absorbance was measured with a 0 × × × portable narrow-bandwidth spectra-radiometer (CI-700, Inc., Vancouver, WA, USA) in a light bench.

4.3. Determination of the Spectral Reflectance, Chl a + b and CarContent Spectral reflectance was measured from the same leaves as ChlF measurement, using CI-700spectra-radiometer (CID Inc., Vancouver, WA, USA). Photochemical reflectance index (PRI) was calculated from the reflectance spectrum as (R531 R570)/(R531 + R570) for assessing xanthophyll cycle − pigments [29]. Plants 2020, 9, 960 11 of 13

Chla + b and Car contents in mature, healthy, and fully expanded middle to upper leaves were determined using methods described previously [30]. In brief, leaf discs were excised using a standard hole punch, immediately sealed in pre-labeled aluminum envelopes, and placed in liquid nitrogen. Tissues were stored at 80 C until analysis, and then extracted in a solvent mixture of , − ◦ , and water (80:15:5, v/v/v) at 4 C overnight. The mixture was centrifugedat 13,000 g for ◦ × 10 min, and the supernatants were then measured by a spectrophotometer (U-2000, Hitachi, Tokyo, Japan) to determine the absorbance of Chla and Chlb in acetone at 663 and 647 nm, respectively. The concentration ofChla, Chlb, and Car was calculated using the following equations:

Chla = 0.01373 OD663 0.000897 OD537 0.003046 OD647, × − × − × Chlb = 0.02405 OD647 0.004359 OD537 0.005507 OD663, × − × − × Car = [OD470 (17.1 (Chla + Chlb) 9.479 Ant)]/119.26. − × − × 4.4. Statistical Analysis All experiments were arranged in a completely randomized design. All parameters were subjected to a one-way analysis of variance (ANOVA) with a significance level of p 0.05 using CoStatstatistical ≤ software (Cohort Berkeley, Monterey, CA, USA). Regression analyses were used to examine relationships among Chla + b and Car. In addition, model datasets were based on at least 36 leaves from each PPFD level and ChlF parameters were calculated using Chla + b data from the model validation datasets. Several models were tested, including the linear and non-linear regression models being selected forthe interpretation of the relationship between ChlF parameters and PPFD. All models were evaluated for goodness of fit by the graphical analysis of residuals and by computing correlation coefficients (r). Each experiment was performed twice independently with a randomized design for the growth environment, sampling day, and ChlF analyses.

5. Conclusions There are many leaf color cultivars in Chinese kale, including green, yellow-green, and dark-green and ChlF components and PRI were used to indirectly measure the different functional levels of photosynthesis on these leaves. We showed that different cultivars of Chinese kale displayed variations in their photosynthetic apparatus associated with Chla + b and Car contents in a PPFD response, in that yellow-green cultivars displayed remarkably lower Chla + bcontents under all PPFD and fertilizer treatments. The relationships of ChlF and PRI parameters with Chl a + b contents under PPFD variations were established and can be used to improve the photosynthetic productivityand provide for eco-physiological research in Chinese kalespecies. Yield and cost are the two most important criteria in agricultural production where environmental factor optimization is concerned. The final goal of our project is to develop a new light irradiance apparatus optimized forChinese kale production in plant factories. This study provides a better understanding of the photosynthetic characteristics of Chinese kalefor promoting the rapid, large-scale, and effective management and cultivation of these plants. These results also provide deeper insight into the interception of light by photosynthetic and photo-protective pigments as a function of light intensity conditions, which is important for plant , as well as the knowledge-driven selection of light irradiances for non-invasive pigment estimations.

Author Contributions: J.-H.W. conceived and designed the experiments; M.-Y.H. and F.-C.S. performed the experiments and analyzed the data; K.-H.L. wrote the paper. All authors approved the final manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Plants 2020, 9, 960 12 of 13

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