The impact of abiotic factors on chlorophyll carotenoid

The impact of abiotic factors on chlorophyll & carotenoid phytochemicals in specialty crops Dean Kopsell Plant Sciences Department The University of Tennessee Knoxville, TN

Presentation Outline �Carotenoids and Chlorophylls in Plants/Foods �Physiological & Nutritional Significance �Impacts of environmental growing conditions: �Light �Fertility �Air temperature �Hormone applications �Herbicide applications �Closing Remarks

Carotenoids in Plants �Bound with Chlorophyll to form LHCs � -carotene in PSI and Lutein in PSII �Xanthophyll cycle functions • • Zeaxanthin/antheraxanthin cycling. Dissipation of excitation energy. – Demmig-Adams et. al. , 1996 �The role of carotenoids in PS • • Light harvesting and photoprotection. Quenching of 3 Chl, singlet 1 O 2, and superoxides. – Frank and Cogdell, 1996

Light-Harvesting Complex Tight Chl a–carotenoid The Vio binding site. Vio (orange) interaction in LHC-II. Chl 5 and sits in a pocket on the monomer Lut 2 are in extensive van der interface with one end exposed to Waals contact through their the lipid bilayer. coplanar systems. Image taken from http: //www. nature. com/emboj/journal/v 24/n 5/fig_tab/7600585 f 5. htm

Carotenoids in Plants �Importance of plant-based carotenoids �Light-harvesting as accessory pigments �Xanthophyll cycle functions �Indicate fruit maturation for dispersal/vectors �Sex-related colorations in animals/insects �Serve anti-oxidant functions �Convey important health properties �Aromatic carotenoids used in perfumes

Chlorophylls in Plants �Importance of plant-based chlorophylls �Light-harvesting pigments in PS. �Evidence of health values associated w/ chlorophylls. �Cancer prevention, anti-mutagenic activity, and induction of apoptosis in tumor cells. �May reduce intestinal absorption of potentially harmful chemical mutagens and carcinogens as detoxifying agents.

Mevalonate Pathway (MVA) Methylerythritol phosphoate Pathway (MEP) Taken from: www. plantphysiol. org/cgi/content-nw/full/130/3/1079/F 1

Enzymatic reactions throughout the pathway are depicted using solid arrows accompanied by the enzyme abbreviations in capitalics. Enzymes abbreviated as: PSY, phyotene synthase; PDS, phytoene desaturase; Z-ISO, ξ-caroene isomerase; ZDS, ξ-carotene desaturase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; HYD, carotene hydroxylase (both β-ring and ε-ring hydroxylases); ZEP, zeaxanthin epoxydase; VDE, violaxanthin de-epoxydase.

Carotenoids in Foods

Carotenoids in Plants �Yellow, red, orange…natural pigments �Colorations for birds and insects �Plants are sources for dietary carotenoids � 600+ identified; 50 -60 in typical diet �Carotenes and xanthophyll carotenoids �Chemoprevention and antioxidant functions �Leafy greens and colored vegetables � Roles as PS accessory pigments � Develop with maturation of fruits

Health Benefits �Conjugated carbon-carbon double bond system permit quenching of 1 O 2. �In vivo antioxidant activity determined by carotenoid structure & concentration. � Localization of carotenoid molecules influence ability to encounter & scavenge free radicals. �Carotenoids are potent biological quenchers of reactive oxygen species � Susceptibility of 5, 6 and 5’, 6’ double bonds in their cyclic end groups to undergo epoxidation with 1 O 2.

Carotenoid carbon positions and structure of zeaxanthin, antheraxanthin, violaxanthin, lutein, and 5, 6 -epoxylutein. Conjugated double bonds at 5, 6 and 5’, 6’ positions are highly effective at quenching singlet oxygen.

Health Benefits �Carotenoids exhibit cis-trans isomerization…both isomeric groups found in vegetable crops. �All-trans carotenoids in plants are susceptible to photo-, thermal-, and chemical-isomerization. �Cis-trans isomers differ in their intestinal absorption in humans. �Human blood plasma contains mostly all-trans carotenoids, but some carotenoids can be found as high as 50% in the cis form. �Postharvest/processing activities impact isomerization.

Commodity Carotenoids Identified Beans, green all-trans -carotene, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans lutein epoxide, 9’-cis neoxanthin, neolutein, all-trans violaxanthin, all-trans zeaxanthin, 9 -cis zeaxanthin, 13 -cis zeaxanthin Broccoli all-trans -carotene, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans and cis lutein epoxide, neolutein, all-trans neoxanthin, 9’-cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9 -cis zeaxanthin, 13 -cis zeaxanthin Cabbage -carotene, lutein epoxide, neoxanthin, violaxanthin, zeaxanthin Carrot all-trans -carotene, lutein, lycopene Corn -carotene, -cryptoxanthin, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans zeaxanthin, 9 -cis zeaxanthin Kale/collards all-trans -carotene, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans and cis lutein epoxide, neolutein, all-trans neoxanthin, 9’-cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9 -cis zeaxanthin, 13 -cis zeaxanthin Lettuce all-trans -carotene, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans and cis lutein epoxide, neolutein, all-trans neoxanthin, 9’-cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9 -cis zeaxanthin, 13 -cis zeaxanthin Pepper -carotene, -cryptoxanthin, capsanthin, lutein, zeaxanthin Spinach all-trans -carotene, all-trans lutein, 9 -cis lutein, 9’-cis lutein, 13 -cis lutein, all-trans and cis lutein epoxide, neolutein, all-trans neoxanthin, 9’-cis neoxanthin, violaxanthin, all-trans zeaxanthin, 9 -cis zeaxanthin, 13 -cis zeaxanthin Tomato (raw) all-trans -carotene, all-trans lutein, all-trans lycopene, neurosporene, phytofluene, lycopene-5, 6 diol

Impact of environmental growing conditions on pigment concentrations

What impacts pigment values? �Light quantity & quality �Photoperiods �Nutrient fertilizers �Growing air temperatures �Plant growth regulators (hormones) �Herbicides/Fungicides/Insecticides �All are plant growth regulators (PGRs)

Light Quantity & Quality

Light Quantity & Quality �Xanthophyll cycle impacted by light intensity. �Cycle functions to remove excess radiational energy. �Easy to manipulate carotenoids in the cycle. �Control of light quality (different nanometers) by light-emitting diodes (LEDs). �Expose plants to single wavelengths. �Preliminary data reveals interesting impacts.

Xanthophyll Cycle Phytoene Desaturation Reactions Lycopene α-carotene β-carotene Low Irradiance Zeaxanthin Low Irradiance High Irradiance Lutein-5, 6 -epoxide De-epoxydation Epoxydation Low Irradiance High Irradiance Lutein Antheraxanthin Violaxanthin High Irradiance De-epoxydation removes oxygen functions and results in the lengthening of the double bonds. It can occur within minutes. Epoxydation occurs within minutes to hours, but can also take days under stress conditions.

Changing Light Quantity � Possible to manipulate xanthophyll cycle pigment levels through exposure to light. � ZEA selectively deposited in the macula region of the eye to provide photo-protection from harmful UV-light. � Study objective to increase ZEA concentrations in mustard (Brassica juncea L. ‘Florida Broadleaf’) microgreens through exposure to high light just prior to tissue harvest. � Microgreen plants grown under 275 µmol/m 2/sec. Light intensity was increased to 463 µmol/m 2/sec. Plants were harvested after 3 days of exposure. � Microgreens measured for carotenoid pigments, especially the xanthophyll cycle pigment of ZEA.

Changing Light Quantity � The xanthophyll cycle in higher plants controls the energy dissipation of excess light excitation through reversible deepoxidation and epoxidation of zeaxanthin, antheraxanthin, and violaxanthin. VDE = violaxanthin de-epoxidase; ZEP = zeaxanthin epoxidase. Adapted from Latowski et al. , 2004 and Demmig-Adams & Adams, 1996.

Changing Light Quantity Mean valuesa for chlorophyll shoot tissue pigments (mg/100 g Fresh Weight) in mustard (Brassica juncea L. ‘Florida Broadleaf’) microgreen plants grown under light treatment conditions of 275 µmol/m 2/sec or light treatment conditions of 463 µmol/m 2/sec just prior to harvest. Shoot tissue pigment 275 µmol/m 2/sec 463 µmol/m 2/sec light treatment Significanceb mg/100 g Fresh Weight Chlorophyll a 9. 83 2. 42 6. 08 1. 49 P 0. 001 Chlorophyll b 10. 44 1. 89 8. 55 0. 97 P 0. 01 Total Chlorophyll 20. 28 4. 01 14. 64 1. 95 P 0. 001 a to b ratio 0. 94 0. 16 0. 72 0. 19 P 0. 01 Mean values standard deviation represent two complete experimental runs, 12 replications of composite samples of microgreen plants for each light treatment per run. b Significance based on paired t-test from exposure of microgreens to 463 µmol/m 2/sec light treatment for 36 hours prior to harvest. a

Changing Light Quantity Mean valuesa for carotenoid shoot tissue pigments (mg/100 g Fresh Weight) in mustard (Brassica juncea L. ‘Florida Broadleaf’) microgreen plants grown under light treatment conditions of 275 µmol/m 2/sec or light treatment conditions of 463 µmol/m 2/sec just prior to harvest. Shoot tissue pigment 275 µmol/m 2/sec 463 µmol/m 2/sec light treatment Significanceb mg/100 g Fresh Weight β-carotene 1. 65 0. 27 1. 37 0. 21 P 0. 01 Lutein 3. 44 0. 56 3. 13 0. 44 ns Zeaxanthin 0. 03 0. 01 0. 07 0. 01 P 0. 001 Antheraxanthin 0. 16 0. 04 0. 24 0. 05 P 0. 01 Violaxanthin 0. 76 0. 18 0. 64 0. 13 ns Neoxanthin 0. 99 0. 19 0. 69 0. 14 P 0. 001 Mean values standard deviation represent two complete experimental runs, 12 replications of composite samples of microgreen plants for each light treatment per run. b ZEA+ANT/ZEA+ANT+VIO = zeaxanthin + antherazanthin/ zeaxanthin + antheraxanthin + violaxanthin. c Significance based on paired t-test from exposure of microgreens to 463 µmol/m 2/sec light treatment for 36 hours prior to harvest. ns = non-significant. a

Blue-light LED & “baby” kale �Measure the impact of blue light on “baby” kale pigments. �‘Dwarf Siberian” kale grown in growth chamber environment. �Day/night temps of 24°C/20°C � 16 hr photoperiod at 250 mol·m-2·sec-1. �Light treatments: �Fluorescent/Incandescent light �LED 20% Blue (455 -470) and 80% Red (627 -630). �LED 5% Blue (455 -470) and 95% Red (627 -630). �Harvested at 30 days old.

Kale Fluorescence Parameters Normalized Chl Fluorescence Yield Control 5% Blue Light 20% Blue Light Control NPQ 1 0. 8 0. 6 0. 4 0. 2 5% Blue NPQ 0 NPQ QY_max Higher NPQ indicative of higher light stress. 20% Blue NPQ

Leaf Tissue Xanthophyll Cycle Control 5% Blue Light 20% Blue Light 2. 5 2 1. 5 1 0. 5 0 Zeaxanthin Antheraxanthin Violxanthin

Leaf Tissue Xanthophyll Cycle Control 5% Blue Light 20% Blue Light 2. 5 2 1. 5 1 0. 5 0 Zeaxanthin Antheraxanthin Violxanthin

Results from Baby Kale Study �LED lighting resulted in a less stressful environment. �Higher NPQ (indicates stress) under control treatment. �Leaf tissue xanthophyll cycle pigments complete story: �Higher ZEA concentrations under control. �LED treatments caused higher VIO concentrations. �Further illustrate eco-physiological responses. �Horticultural/nutritional advantages to proper management.

Light Intensity Influences Kale leaf tissue pigment concentrations Mean valuez of pigment accumulations in the leaf tissues of ‘Winterbor’ kale (Brassica oleracea L. ) grown under increasing irradiance levels in nutrient solution culture. mol m-2 sec-1 Lutein -carotene Chl ax Chl bx Pigment Concentration (mg· 100 g-1 FM) 125 200 335 460 620 9. 1 1. 4 12. 0 1. 6 15. 1 1. 4 12. 0 0. 6 12. 7 0. 5 5. 7 0. 9 8. 1 1. 1 11. 1 1. 2 8. 6 0. 5 9. 3 0. 4 145. 0 23. 1 184. 4 26. 0 247. 3 10. 0 211. 4 13. 0 216. 3 18. 2 35. 1 6. 4 47. 2 6. 7 59. 0 2. 2 55. 1 1. 5 55. 2 4. 2 ns * * ** ** ** Contrastsy L Q Mean composition of sampled leaf tissue of 6 replications, 8 plants each standard deviation. Significance for linear (L) and quadratic (Q) orthogonal contrasts. ns, *, *** Non-significant or significance at P 0. 05, P 0. 01, P 0. 001, respectively. z y Lefsrud et al. , 2006. Physiologia Plantarium 127(4): 624 -631.

Changing Wavelengths �Photosynthetic Active Radiation (PAR) � 400 to 700 nm LED Array 400 nm ultraviolet 440 nm royal blue 520 nm green 640 nm red 730 nm near Infrared Air temperature of 20 ± 1 C and 15. 2, 253. 3, 6. 5, 10. 6, and 6. 9 µmol m -2 s-1 for 730, 640, 525, 440, and 400 nm, respectively.

Changing Wavelengths �Light emitting diodes (LED) for plant production is a new field of research. �Next new technology in GH lighting. �Kale plants (Brassica oleracea L. var. acephala D. C. ) were grown under specific LED wavelength treatments of 730, 640, 525, 440 and 400 nm to determine changes in the accumulation of chlorophylls, carotenoids, and glucosinolates. Lefsrud, et al. , 2008. Hort. Science 43(7): 2243 -2244.

730 nm 640 nm 525 nm 440 nm 400 nm

Changing Wavelengths Mean pigment (mg 100 g-1 fresh mass) and glucosinolate (mg 100 g-1 dry mass) concentrationsz in the leaf tissues of ‘Winterbor’ kale grown under specific wavelength using LEDs. Pigment Glucosinolate (mg 100 g-1 FM) (mg 100 g-1 DM) Lutein -carotene Chl ay Chl by Sinigrin 730 6. 9 1. 0 2. 7 0. 4 31. 1 10. 1 16. 0 21. 7 640 11. 2 0. 4 3. 7 0. 4 85. 7 7. 2 66. 2 2. 8 32. 0 16. 6 525 7. 8 0. 8 3. 3 0. 4 51. 2 10. 5 31. 8 6. 5 0. 8 440 9. 8 0. 7 4. 0 0. 4 63. 8 4. 3 33. 9 5. 1 ndy 400 8. 1 1. 1 3. 4 0. 3 57. 9 3. 7 28. 8 3. 9 ndy Wavelength (nm) z y Mean composition of sampled leaf tissue of three replications and one plant standard error. Chl a = chlorophyll a; Chl b = chlorophyll b, nd = non detected.

Changing Wavelengths �ZEA hypothesized as blue-light receptor, so can ZEA levels be influenced by Blue (470 nm) LED light? �Sprouting Broccoli grown in controlled environment chamber at 24 -h photoperiod under an average light intensity of 350 µmols/m 2/sec 1 from light-emitting diodes of 470 nm and 627 nm. �Control chamber [350 µmol m 2 sec 1 from 470 nm (Blue) and 627 nm (Red)] �Treatment chamber [41 µmol m 2 sec 1 from 470 nm (Blue)]. �Plants grown for 5 -days under treatments.

350 mol/m 2/sec 627 & 470 nm 41 mol/m 2/sec 470 nm

Changing Wavelengths Mean valuesa for chlorophyll shoot tissue pigments (mg/100 g Fresh Weight) in Sprouting Broccoli (Brassica oleracea L. ) grown under light-emitting diode (LED) treatment conditions of red (627 nm) and blue (470 nm) at 350 µmol/m 2/sec, or a 5 -day pre -harvest light treatment of only blue (470 nm) at an average of 41 µmol/m 2/sec. Shoot tissue pigment Red and Blue LED light treatment (350 µmol/m 2/sec) Blue LED light treatment (41 µmol/m 2/sec) Significanceb mg/100 g Fresh Weight Chlorophyll a 40. 80 2. 90 36. 75 2. 90 ns Chlorophyll b 16. 40 0. 95 13. 50 0. 95 P 0. 04 Total Chlorophyll 57. 20 3. 81 50. 25 3. 81 ns a to b ratio 2. 47 0. 06 2. 71 0. 06 P 0. 01 Mean values standard error represent three complete experimental runs, 8 replications of composite samples of sprouting plants for each light treatment per run. b Significance based on paired t-tests. a

Changing Wavelengths Mean valuesa for carotenoid shoot tissue pigments (mg/100 g Fresh Weight) in Sprouting Broccoli (Brassica oleracea L. ) grown under light-emitting diode (LED) treatment conditions of red (627 nm) and blue (470 nm) at 350 µmol/m 2/sec, or a 5 -day pre -harvest light treatment of only blue (470 nm) at an average of 41 µmol/m 2/sec. Shoot tissue pigment Red and Blue LED light treatment (350 µmol/m 2/sec) Blue LED light treatment (41 µmol/m 2/sec) Significanceb mg/100 g Fresh Weight β-carotene 2. 17 0. 16 2. 62 0. 16 P 0. 05 Lutein 4. 31 0. 27 3. 92 0. 27 ns Zeaxanthin 0. 04 0. 01 0. 03 0. 01 ns Antheraxanthin 0. 35 0. 03 0. 34 0. 03 ns Violaxanthin 0. 99 0. 10 1. 40 0. 10 P 0. 01 Neoxanthin 1. 22 0. 08 1. 02 0. 08 ns Mean values standard error represent three complete experimental runs, 8 replications of composite samples of sprouting plants for each light treatment per run. b Significance based on paired t-tests. a

Changing Light Quality Light quality can affect TOMATO carotenoid levels �Fruit lycopene concentrations can be increased with higher light intensity, under favorable temps. �Red Light can enhance lycopene synthesis in detached fruit. �Far-Red Light stops lycopene synthesis. �Indicates a Phytochrome involvement. � Thomas and Jen, 1975; Alba et al. , 2000.

Nitrogen Fertility

Nitrogen & Carotenoids �Will Nitrogen (N) affect plant carotenoids? � N-form/N-levels can affect plant growth. � Part of the chlorophyll molecule…photosynthesis. � Chlorophylls correlate highly with carotenoids. �N fertilization can affect plant metabolism. � Fertilization can be controlled by producers. �Will N and N-form affect phyto-nutrients? � Specifically the carotenoid compounds.

Nitrogen & Carotenoids �Hydroponic culture �Different N levels �NH 4+ : NO 3 - ratios �Leafy vegetables �HPLC analysis Kopsell, et al. , 2007. Journal of the Science of Food and Agriculture 87(5): 900 -907.

Changing N Levels Nitrogen level affected KALE lutein levels No significant trends Linear trends (P = 0. 001) mg per g Dry Wt. mg per 100 g Fresh Wt. ‘Winterbor’ Kale 6 13 26 52 105 6 mg Nitrogen per Liter 13 26 52 105

Changing N Levels Nitrogen level affected SPINACH lutein levels Quadratic trends (P =0. 05) Linear trends (P =0. 001) mg per g Dry Wt. mg per 100 g Fresh Wt. ‘Springer’ Spinach 13 26 52 105 13 mg Nitrogen per Liter 26 52 105

Changing N Levels Nitrogen level affected PARSLEY lutein levels mg per 100 g Fresh Wt. Chenard, et al. , 2005. Journal of Plant Nutrition 28(2): 285 -297. Highly significant LINEAR 6. 5 13 26 52 mg Nitrogen per Liter 105 increases

Changing N Levels Nitrogen level can affect TOMATO carotenoid levels �Tomato cv. ‘Moneymaker’ � Aziz, 1968. �Grown hydroponically at 1. 0, 12. 9, and 15. 8 meq N/L �Lycopene concentrations were 68, 44, and 38 mg/kg, respectively. �For color development in tomato, nitrogen supply should be as low as possible during maturation.

NH 4+-N vs. NO 3 --N N as 100% NH 4+ N as 50% NH 4+ N as 0% NH 4+

Changing N Form mg Lutein per 100 g FW Nitrogen form influenced LUTEIN levels in kale. Linear P=0. 033 Linear P=0. 001 NS 100% 75% 50% 25% 0% % Ammonium-N in solution 100% NO 3 -

Changing N Form mg -carotene per 100 g FW Nitrogen form influenced -carotene levels in kale. Linear P=0. 003 Linear P=0. 001 Linear P=0. 01 100% 75% 50% 25% 0% % Ammonium-N in solution 100% NO 3 -

Nitrogen & Carotenoids Research Conclusions • N form and N level affected biomass. • Changing the level of N present: • Increases in L and BC in leafy crops. • Decreases lycopene in tomato fruit. • Changing the form of N present: • Increases in L and BC with more NO 3 -. • N influences specialty crop carotenoids. Nitrogen is controllable by producers.

Growing Air Temperature

Air Temperature 15°C 20°C 25°C 30°C

Growing Temperature influences Kale leaf tissue carotenoids Linear trend (P =0. 001) mg per 100 g Fresh Wt. Linear trend (P =0. 001) Air temperature ( C) Lefsrud, et al. , 2005. Hort. Science 40(7): 2026 -2030.

Changing Temperatures Temperature can affect TOMATO carotenoid levels Carotene content (mg/kg fresh mass) 20 C 30 C Phytoene Phytofluene β-Carotene ξ-carotene γ-carotene Neurosporene Lycopene 18. 4 10. 5 7. 5 1. 0 0. 5 0. 4 40. 5 5. 6 0. 5 6. 6 0. 1 1. 1 Total carotenoids 78. 9 14. 1 Carotene Baqar and Lee, 1978

Plant Hormones Exogenous apo-carotenoid applications

Carotenoid cleavage dioxygenases Ohmiya, 2009

Exogenous applications Materials & Methods �Foliar applications of apo-carotenoid(s). �Rates of 0, 100, 200, and 400 mg per Liter. �Sprayed at weekly intervals from bud initiation to mature green fruit stage.

Fruit Tissue β-carotene mg per 100 g fresh weight 72 % increase from control Foliar Apo -carotenoid (ppm)

Fruit Tissue Lutein mg per 100 g fresh weight 31 % increase from control Foliar Apo -carotenoid (ppm)

mg per 100 g fresh weight Fruit Tissue Lycopene Foliar Apo -carotenoid (ppm)

Exogenous applications Materials & Methods �Soil drench applications of apo-carotenoid(s). �Apo-1, Apo-2, Apo-1+2, and control treatments. �Applied to carrot plants over the growth cycle.

Root Tissue -carotene mg per 100 g fresh weight 37 % increase from control 9 8 7 6 5 4 3 2 1 0 Ap-1 Drenc. Ap-2 h Ap Ap-1+2 o-caroteno id Control s

Root Tissue -carotene mg per 100 g fresh weight 23 % increase from control 12 10 8 6 4 2 0 Ap-1 Drenc. Ap-2 h Ap Ap-1+2 o-caroteno id Control s

Phytoene is a colorless carotenoid with extremely high anti-oxidant properties. Root Tissue phytoene mg per 100 g fresh weight 100 % increase from control 0. 6 0. 5 0. 4 0. 3 0. 2 0. 1 0 Ap-1 Drenc. Ap-2 h Ap Ap-1+2 o-caroteno id Control s

Exogenous applications Results & Discussion �Foliar apps impacted β-carotene in tomato. �Soil drench apps impacted both carotenes in carrot. �May be feedback or impact on CCD functions. �Physiological, not molecular, approach to enhancement.

Carotenoids and ABA Biosynthesis

Applying ABA to Plants �‘Micro. Tina’ tomatoes (dwarf variety developed at UF). �Grown in nutrient solutions. � Full-strength Hoagland’s solutions used. �Selected four ABA concentrations for study. � 0. 0, 0. 4, 2. 0, 10. 0 and 50. 0 mg ABA/L. � Applied to nutrient solutions. �Treatments applied w/no flowers, but buds were present. �Red ripe fruits harvested & measured for carotenoids and carbohydrates.

Visual Observations � 24 hours after ABA treatment initiation: �Visible leaf curling on 50. 0, 10. 0, and 2. 0 mg ABA/L. �No symptoms on 0. 4 mg ABA/L � 10. 0 and 2. 0 mg ABA/L plants out-grew leaf curling within 4 days. �Leaf curl still visible on 50. 0 mg ABA/L trt. �Visible biomass reduction on plants in 50. 0 mg ABA/L.

0. 4 mg ABA/L Control 2. 0 mg ABA/L

10. 0 mg ABA/L Control Drought stress symptoms 50. 0 mg ABA/L

% Change from Control Tomato Fruit Lycopene ABA con centratio ns in Nut rient Sol u tions (m g/L)

% Change from Control Tomato Fruit Lutein ABA con centratio ns in Nut rient Sol utions (m g/L)

% Change from Control Tomato Fruit Beta-carotene ABA con centratio ns in Nut rient Sol u tions (m g/L)

Sink Reduced?

Herbicides

Carotenoid Biosynthesis Chemical Inhibitors Isoprenoid Biosynthesis Shikimate Pathway Tyrosine Methylerythritol phosphoate Pathway (MEP) Pyruvate Clomazone Mevalonate Pathway (MVA) p-hydroxyphenylpyruvate dioxygenase (HPPD) Fosmidomycin acetyl-Co-A Cytosplasm plastoquinone α-carotene Chloroplast Isopentenyl diphosphate (IPP) Carotenoid Biosynthesis CPTA DFPC Lycopene DOXP reductoisomerase 2 -C-methyl-D-erythritol 4 -phopsohate Mesotrione Lycopene cyclase (LCY) DOXP synthase 1 -deoxy-D-xylose 5 -phosphate homogentisate β-carotene + D-glyceraldehyde phosphate Phytoene desaturase (PDS) Zeta-carotene desaturase (ZDS) Phytofluene Norflurazon Dimethyallyl pyrophosphate (DMAPP) Geranyl pyrophosphate (GPP) Phytoene synthase (PSY) Farnesyl pyrophosphate (FPP) Geranylgeranyl pyrophosphate (GGPP)

Carotenoid Biosynthetic Pathway

Phytoene desaturase (PDS) X Mesotrione indirectly blocks PDS activity Carotenoid Biosynthetic Pathway

Labeled for: Field Corn Sweet Corn Popcorn As a CBI, it will impact carotenoids. Company had no data.

Herbicides & Carotenoids �Mesotrione is a herbicide that indirectly inhibits phytoene desaturase in plant tissues, the first step in the carotenoid biosynthesis pathway. � Inhibits the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD), a precursor to plastoquinone and tocopherols. �Research currently underway to determine the efficacy of mesotrione for broadleaf & grassy weed control in managed turfgrass systems. �‘Riviera’ bermudagrass (Cynodon dactylon (L. ) Pers. ) was treated with mesotrione at 0. 28 kg ha-1 or untreated and sampled for tissue pigment concentrations at 0, 3, 7, 14, 21, 28 and 35 days after treatment (DAT).

Leaf blade percent visual whitening (%) and phytoene concentrations (mg 100 g-1 Fresh Weight) in ‘Riviera’ bermudagrass (Cynodon dactylon (L. ) Pers. ) treated with mesotrione at 0. 28 kg ai ha-1 (●) and sampled at 0, 3, 7, 14, 21, 28 and 35 days after treatment. Means pooled from two experimental runs, with error bars indicating standard deviations.

Leaf blade chlorophyll a and chlorophyll b concentrations (mg 100 g-1 Fresh Weight) in ‘Riviera’ bermudagrass (Cynodon dactylon (L. ) Pers. ) treated with mesotrione at 0. 28 kg ai ha-1 (●) or untreated control (○) and sampled at 0, 3, 7, 14, 21, 28 and 35 days after treatment. Means pooled from two experimental runs, with error bars indicating standard deviations.

Leaf blade lutein and β-carotene (mg 100 g-1 Fresh Weight) in ‘Riviera’ bermudagrass (Cynodon dactylon (L. ) Pers. ) treated with mesotrione at 0. 28 kg ai ha-1 (●) or untreated control (○) and sampled at 0, 3, 7, 14, 21, 28 and 35 days after treatment. Means pooled from two experimental runs, with error bars indicating standard deviations.

Leaf blade zeaxanthin and violaxanthin (mg 100 g-1 Fresh Weight) in ‘Riviera’ bermudagrass (Cynodon dactylon (L. ) Pers. ) treated with mesotrione at 0. 28 kg ai ha-1 (●) or untreated control (○) and sampled at 0, 3, 7, 14, 21, 28 and 35 days after treatment. Means pooled from two experimental runs, with error bars indicating standard deviations.

Herbicides & Carotenoids � Mesotrione initially decreased chlorophyll and carotenoid concentrations in bermudagrass after application � Temporary cessation of phytoene desaturase activity resulted in accumulations of phytoene in treated plants. � However, pigments increased beyond untreated levels by the end of the study. � Upon re-greening of leaf tissues, and resurgence of phytoene desaturase activity, a greater flux of phytoene into the biosynthetic pathway resulted in greater accumulations of downstream carotenoids. � Due to the antioxidant activity of carotenoid pigments, these increases may impact bermudagrass stress tolerance.

Carotenoid Recovery �Turf was susceptible to the mesotrione. � Displayed visual tissue bleaching. �Turf recovered from bleaching with higher levels of carotenoids. �Remember product is labeled for sweet corn. What is happening to the carotenoid in the kernels following applications of mesotrione?

Sweet Corn �Significant impact on agricultural economy. � 94, 860 hectares (234, 404 acres) grown in ‘ 07. �Total production value of $625 million. �Flavor & nutritional attributes. �One of only a few vegetables sources of zeaxanthin carotenoids. �Large acreages sprayed with mesotrione.

Sweet Corn Study �Evaluate impacts of mesotrione on corn. � Sweet corn cultivars used were: �‘Merit’- yellow-kernel sensitive genotype �‘Temptation’- bicolor tolerant genotype �’Incredible’- yellow-kernel moderately sensitive genotype �Post-emergence applications of mesotrione & atrazine to young corn plants.

Sweet Corn Study �Herbicide treatments (following lable): � untreated control � mesotrione at 105 g ai/ha EPOST � mesotrione at 105 + atrazine at 560 g ai/ha EPOST � mesotrione at 105 g ai/ha LPOST � mesotrione at 105 + atrazine at 560 g ai/ha LPOST �Visual rating taken during season, then measured kernels for carotenoids at harvest. Kopsell, et al. , 2009. Journal of Agricultural and Food Chemistry 57(14): 6362 -6368.

Sweet Corn Study EPOST to corn 5 -10 cm tall. May 17, 2008. LPOST to corn 15 -20 cm tall. May 30, 2008. Label stated corn may be sprayed up to 75 cm tall (8 th leaf stage). Also states bleaching may occur on some sweet corn hybrids.

Cultivars 14 days after EPOST treatment applications ‘Merit’ sensitive ‘Incredible’ moderatly sensitive ‘Temptation’ tolerant

Kernel Carotenoids ‘Merit’ – sensitive genotype treatment timing Untreated Lutein % change Zeaxanthin 0. 632 % change 0. 509 Meso EPOST 0. 681 + 7. 8 0. 570 + 12. 0 Meso + Atrazine EPOST 0. 706 + 11. 7 0. 583 + 12. 7 Atrazine EPOST 0. 692 + 9. 5 0. 564 + 10. 8 Meso LPOST 0. 577 - 8. 7 0. 409 - 3. 7 Meso + Atrazine LPOST 0. 672 + 6. 3 0. 582 + 14. 8 Atrazine LPOST 0. 664 + 5. 1 0. 550 + 8. 1 % change from untreated check (control treatment)

Kernel Carotenoids ‘Incredible’ – moderately sensitive genotype treatment timing Untreated Lutein % change Zeaxanthin 0. 328 % change 0. 448 Meso EPOST 0. 307 - 6. 4 0. 415 - 7. 4 Meso + Atrazine EPOST 0. 399 + 21. 6 0. 498 + 16. 4 Atrazine EPOST 0. 353 + 7. 6 0. 447 - 0. 0 Meso LPOST 0. 343 + 4. 6 0. 454 + 1. 3 Meso + Atrazine LPOST 0. 340 + 3. 7 0. 436 - 2. 8 Atrazine LPOST 0. 347 + 6. 0 0. 454 + 1. 3 % change from untreated check (control treatment)

Kernel Carotenoids ‘Temptation’ – tolerant genotype treatment timing Untreated Lutein % change Zeaxanthin 0. 359 % change 0. 203 Meso EPOST 0. 381 + 6. 1 0. 212 + 4. 4 Meso + Atrazine EPOST 0. 335 - 6. 7 0. 183 - 9. 9 Atrazine EPOST 0. 346 - 3. 6 0. 198 - 2. 5 Meso LPOST 0. 355 - 1. 1 0. 193 - 4. 9 Meso + Atrazine LPOST 0. 351 - 2. 2 0. 199 - 2. 0 Atrazine LPOST 0. 346 - 3. 6 0. 194 - 4. 4 % change from untreated check (control treatment)

Study Results �Genetic variations in sensitivity in hybrids. � Some corn bleached and then recovered. �Mesotrione impacted kernel carotenoids. � EPOST showed greater impacts. �Mesotrione + atrazine had the greatest impacts. �Greater pools of carotenoids post-recovery. �Carotenoids are stress-response compounds. �First evidence of a herbicide impacting nutritional quality of a vegetable crop.

Closing Remarks �Many abiotic/environmental factors can impact plant growth & development. �When these factors impact physiology, they also impact production of 2° metabolites (phyto-chemicals). �Management of abiotic factors critical for improving nutritional quality. �Manipulation of specific metabolic pathways are possible to improve nutritional values. �Data is showing the ability to regulate nutritionally important compounds through cultural management.

Thank you…

Extra Slides for Students. More information of carotenoid physiology in plants.

Carotenoid Biochemistry �The carotenoid biosynthetic pathway elucidated in the mid 1960 s. �Carotenoids produced in the plastids and derived from isopentenyl diphosphate (IPP). �First step in biosynthesis - IPP is isomerized to dimethylallyl diphosphate (DMAPP), then becomes C 20 geranyl diphosphate (GGPP). � The enzyme GGPP synthase catalyses formation of GGPP from IPP and DMAPP. �First step unique to carotenoid biosynthesis is the condensation of 2 molecules of GGPP to form C 40 carotenoid phytoene, via phytoene synthase.

Cunningham and Gantt, 1998 Annu. Rev. Plant Physiol. Plant Biol.

Carotenoid Biochemistry �Phytoene desaturase and -carotene desaturase, make conversions of phytoene to lycopene. �These desaturase enzymes create the chromophore of carotenoid pigments and change the colorless phytoene into the pink-colored lycopene. �Pathway branches at cyclization of lycopene to produce carotenoids with either 2 -rings or 1 -ring & 1 -ring. �Pathway advances with additions of oxygen moieties, which convert hydrocarbon - and -carotenes into subgroup referred to as the xanthophylls.

Cunningham and Gantt, 1998 Annu. Rev. Plant Physiol. Plant Biol.

Escherichia coli strain TOP 10 genetically engineered to accumulate the carotenoid indicated on the petri dish. Cunningham and Gantt, 1998 Annu. Rev. Plant Physiol. Plant Biol.

Carotenoid Biochemistry �In the pigment-protein complexes of photosynthetic organisms, the all-trans configurations of carotenoids are the major component of the LHCs. �All-trans carotenoids provide efficient singlet-energy transfer to chlorophyll molecules, and thus participate in light-harvesting. �The 15 -cis carotenoids show preference for isomerization towards the all-trans configurations upon excitation. �Better suited for photo-protective functions.

Carotenoid Isomerization Structures of some common all -trans and cis carotenoid isomers found in fruit and vegetable crops. The occurrence and properties of carotenoid isomers in food crops/food stuff are affected by post-harvest processing and can influence intestinal absorption.

Carotenoid Isomerization Structures of some common all beta-trans. All-trans and cis carotenoid carotene excreted isomers found in fruit andless vegetable The thancrops. cis-forms. occurrence and properties of carotenoid isomers in food crops/food stuff are affected by post-harvest processing and can influence intestinal absorption. Cis-forms of lycopene absorbed in higher concentrations.

Changing Light Quantity � Carotenoids are important photo-protectant & light-harvesting pigments within the PS apparatus. � Eco-physiology study using bentgrass (Agrostis stolonifera L. ) as a model. � ‘Crenshaw’ creeping bentgrass plants acclimated for 7 -days to relative high [47. 9 mol day-1 photosynthetically active radiation (PAR)] or low irradiance (4. 7 mol day-1 PAR). � After acclimation period, plants were transferred from high to low (low irradiance adaptability) irradiance and low to high (high radiance adaptability). � Clippings were harvested at 0, 24, 72, and 168 h after acclimation period & measured for carotenoid concentrations.

Changing Light Quantity Changes in the dark red pie sections represent impacts on the xanthophyll cycle in the bentgrass plants. Taken from Mc. Elroy J. S. et al. 2006. Crop Sci. 46(6): 2606 -2612.