b 2011 Society of Chemical Industry ∗ c a ) and N fertility (0, 72, 144, and + / − 6–10 Plant compounds acting as antioxidants and 5–9 Lactuca sativa Robert W Mullen, a The Ohio State UniversityWooster, – OH, OARDC, 44691 USA Environmental and Natural Resources, The Ohio State University, Horticulture and Crop Science,USA Columbus, OH, 43210 The OhioEngineering, State Wooster, OH, 44691 USA University – OARDC, Food, Agricultural, and Biological Correspondence to: Matthew D Kleinhenz, The OhioHorticulture State and University Crop – Science, OARDC, 1680 Madison Ave.,E-mail: Wooster, [email protected] OH, 44691 USA. The Ohio State University –OH, OARDC, 44691 USA Horticulture and Crop Science, Wooster, The causal mechanisms underlying the protective effects of ∗ c a d b from many disciplines. Beyond providing essentialand nutrients, vegetable fruit intakehuman appears health factors, including to threats of impact stroke andand cancer, other short- heart disease. and long-term fruit and vegetable consumption are not all clear; however, there radical scavengers is one of several proposedplant-based mechanisms protection. for this and Matthew D Kleinhenz d Temperature, N or S latitude). ◦ 1,2 40 > Joseph C Scheerens, a Peter P Ling c : 116–124 www.soci.org 92 Interest in plant biomass accumulation and ). Root-zone heating increased but withholding N decreased biomass accumulation in both years. Low N supplies 2012; 1 3–6 − seasonal production; human nutrition; antioxidant; root-zone; fertility;

2011 Society of Chemical Industry Human health benefits associated with fruit and vegetable c composition – specifically, thesystems efficiency convert with naturala which and selected suite often cropping of compounds diminishinghuman – nutrition, is resources energy fueled production and in into other part arenas. Overall,identification by the their of roles microclimates in conduciveof to crops abundant with harvests anmore idealized effectively deploy make-up agricultural lags systemsand behind enhancement in the of the human interest maintenance well to being,availability of e.g. nutritionally as dense influenced products. by This the particularly knowledge wide gap when is considering the design ofoperating outdoor systems in areas with distinct seasons (e.g. J Sci Food Agric INTRODUCTION Modern horticultural systems relyenvironments heavily immediately on the surrounding ability crops. to alter 576 mg day

Abstract BACKGROUND: Understanding theconditions effects during cool of seasons ofnitrogen temperature temperate (N) climates, and fertility is on nitrogen important. thesystem Here, was accumulation levels employed. we and document on composition the of key impact fall- of variables, root-zone and heating spring-grown particularlyRESULTS: and lettuce Direct-seeded under plots biomass. containing A field a novel,in uniform, scalable semi-solid, 2009 field and and nearly stable 2010; rooting each medium were contained established one outdoors of eight combinations of root-zone heating ( lettuce Mark A Bennett, of fall- and spring-grown, high-density leaf Natalie R Bumgarner, yield and secondary metabolite concentration Root-zone temperature and nitrogen affect the (wileyonlinelibrary.com) DOI 10.1002/jsfa.4549 Research Article Received: 22 December 2010 Revised: 22 April 2011 Accepted: 20 May 2011 Published online in Wiley Online Library: 12 August 2011 light and mineral nutrientmanaged levels microclimate are among components.the the Together, most production intensely they andwith influence composition the ofwith formation crop the of biomass synthesis,compounds. beginning primary deposition metabolites and and breakdown concluding of secondary were also associatedchlorophyll a with and vitamin greater C levels anthocyanin tracked root-zone and temperature and total NCONCLUSIONS: fertility Experimentally antioxidant more imposed closely root-zone in power temperature 2009 andspring-grown and but N 2010, lettuce levels respectively. lower influenced tissue. the Ambient amount N conditions,variables and measured. properties however, and Collectively, of dictated the fall- phosphorus results which and point of towith levels. the these respect potential to factors Tissue for supplying gains exerted human in the nutritional system units. sustainability greatest and effect productivity, including on the Keywords: consumption are increasingly clear and supported by research

116 117 2 N; longitude: . Here, fertility 1  − 46 C. The operation of ◦ ◦ . Tunnel sides and ends 1 − wileyonlinelibrary.com/jsfa (Brookside Laboratories, Inc., New 3 1.25 cm; Dow Chemical Co., Midland, − main plot × 3 15 m) main plots contained all four 0.36 m . 0.60 m 0 × × ). For both experimental runs, treatments were 4 . 1 2 − × of medium and new medium was used in 2009 and W). The study was located in an outdoor gravel-bed 6  . 3 55 ◦ (OARDC) in Wooster, OH, USA (latitude: 40 laboratory operated byand the Biological Department Engineering. ofan OARDC Food, Argus is Agricultural TitanColumbia, a Nutrient Canada) beta which Delivery testing wasTitan System used site system (White throughout for is the Rock,delivery study. capable regimens, British The of inmulti-feed part executing system has through been highly designed single for small-scale tailored experimental and element commercial nutrient dosing. application and The is capablenutrient of solution timed delivery and accurate rates of 0.25–10 L min randomized N fertility(Hummert sub-plots. International, Woven St Louis, plasticstaples to MO, ground the USA) cover main wasRoot-zone plot heated attached frames main plots to with included a contain 12.2heating the m cable automatic inorganic electric (Wrap-On media. Co.,function Bedford at Park, medium IL, temperatures below USA) 23 triggered to treatments were implemented bythe calculating timed and injection of programming stock solutions ateach dilution ratios chosen resulting in fertilitythen level. Valves delivered and eachtargeted separate specifically outdoor irrigation raised mixed lines 30 beds m treatment from at the solution injection the point.system to were All study controlled aspects electronically through site of a the software approximately interface connected nutrient delivery to sensorsvolume, which electrical continuously conductivity monitored (EC),irrigation pH the solution and (fertigation) flow temperature to ensure ofnutrient delivery levels. the of the selected Experimental and growing system design This factorial experiment included two levels of(yes/no) root-zone heating and four levels576 mg of day nitrogen (N) supply (0, 72, 144 and replicated four timesroot-zone and heating and arranged N fertility in functioning asplots, a the main respectively. split-plot and sub- All designbed eight with (0 wood-framed, randomized, raised were secured to thedividers frame (0.15 with m wooden lath. Polystyrene foam MI, USA) separated fertility sub-plots within each mainof plot. A KapMat layer cloth (BFG Supplyon Co., the Burton, sides OH, and USA)and was bottom prevent installed of cross-contamination each between sub-plot NThe fertility to growing treatments. enhance medium drainage contained the(by following volume): two 60% components industrialChardon, grade OH, 20 : USA) 40 silica andconditioner (Profile sand Products, 40% LLC, (Best Buffalo Primera Grove, Sand, IL, Onecontained USA). 50.1% Medium calcinated total pore clay space (32.0% field a air, 18.1% bulk capillary) density with of 1.32 g cm Knoxville, OH, USA). Thea two Bouldin and dry Lawson components modelMcMinnville, were 12 TN, 193 USA). blended mixer Sub-plots in (Bouldin were loaded and with0.04 Lawson, approximately m 81 2010. each cable was governed by an integrated thermostatcontact in with continual the rooting medium. The cable waswith securely zip attached ties to the plastic ground coverprevent to cable assure contact. even heating All and main plots were coveredslitted with polyethylene a low 0.02 tunnel (Hummert mm International) stretched over four wire hoopsof to create approximately partially 0.50 enclosed m aerial volumes and 8,23–25 14–17 13,18–20 2011 Society of Chemical Industry Lactuca sativa c Also, low N levels are reported The latter point is germane to With intervention, agricultural 13 3–9 as phenylalanine ammonia lyase and 12,26–29 : 116–124 92 Still, increases in secondary compound levels Relationships involving N levels and secondary 3,11–13 Likewise, fluctuations in phenolic metabolism 14,15 2012; 18 14,15,21,22 Secondary metabolite levels influence far more than human Antioxidant activity and secondary metabolite composition Root-zone temperature and nitrogen affect the yield and secondary metabolite www.soci.org health, of course.photosynthetic Anthocyanins apparatus aredamage when low thought against temperatures slow photosynthesis, to photo-induced protect oxidative the under low N may bephenyalanine due to by the need phenylalanine forfor ammonia ammonia released the lyase from activity photoprotectionby and/or of low N. photosynthetic systems disrupted chalcone synthase mRNA levels increaseperatures in in response the presence to of low light. tem- may be associated with decreases in primary productivity. J Sci Food Agric EXPERIMENTAL Site and nutrient delivery system The experiment was2010 conducted at in the Ohio duplicate Agricultural runs Research and in Development 2009 Center and Given the influencetemperature) of on abiotic conditions primarythese (e.g. and conditions nutrition, secondary in light, position productivity, agricultural in setting systems healthbeen and will given commerce. to strengthen setting Little their environmentalof attention the conditions year during to with historically portions date low levels has of production. in leafy cropsand are temperature levels. reported to fluctuate with nutrient, light is little doubtsecondary on compounds other (e.g.carotenoids) matters, phenolics, can namely: act vitamin (1) asnumerous, C that antioxidants, widely and many and distributed (2) various plant by and that environmental phenolics often conditions. are profoundly influenced the heightened design ofstruments sustainable of production systems health as andmicroclimate–yield–tissue in- commerce, composition in relationships. partture The provides through litera- insight on managing some of theseanthocyanin relationships. biosynthesis For example, and accumulation arelow up-regulated by temperature, metabolism are only partially characterized but emerging. to increase phenylpropanoid pathway activity or the levels ofway path- products. L.) biomass accumulation andas composition. the Lettuce was experimental chosen cropto because a wide it: range (1) of tolerates environmentalconsumed conditions, and at (2) is responds a harvested and rangecomponent of of developmental stages, acommodity among (3) producers is and healthy consumers, a especially in diet,Midwest, the leading US and (5) (4) has is been includedducted in an indoors previous and outdoors. related increasingly Employing work a con- noveland rooting popular reliable medium nutrient delivery system,regarding we the gained interplay valuable of insight primarylettuce and exposed to secondary historically metabolism under-studied in conditions. productivity can be maintained whenconditions ambient (e.g. seasonal low growing temperature and/or light) otherwiseit. preclude However, too little is knownmicroclimate regarding management the techniques influence on of crop specific biomasslation accumu- and composition during thesealter periods. temperature, While light, tools humidity, exist soil to moisturecomponents and of other the abiotic plant environment,the this work direct focuses modification only of on soiland their temperature separate and and combined nitrogen effects on fertility lettuce ( they may also protectas against low osmotica. temperature stress by acting . 54 38 et al . . 0 0 C) and ± ± ◦ : 116–124 11 52 . . 92 2012; 14 19 092 20 . . 0 0 ± ± 77 63 . . C until being processed in ◦ 80 J Sci Food Agric − 094 11 14 15 . . 0 0 ± ± Cor Root-zone heating ◦ C) until measurements were taken. All ◦ 99 57 C. A 10 g sample of frozen tissue was . . ◦ 20 − 20 No Yes − 053 10 065 14 . . 0 0 ± ± Average above and below surface temperatures ( 17 87 . . 60 cm section of each sub-plot and placing it on ice until × Table 1. Year Above surface2009 Below surface 10 Above surface Below surface standard errors in 2009beds with and or 2010 without the in incorporation of outdoor, root-zone plastic-covered heating cables raised 2010 14 Environmental data Air and soil temperatures wereintervals recorded using continuously Hobo at 15-min ProV2Pocasset, MA, data USA). Each loggers of (Onset thewith main Computer heating a plots Co., was separate equipped covered data with logger a attached radiation20 to cm above shield. the a media Air surface and wooden soil temperature4–5 temperature was stake was cm recorded below and recorded the surface.air temperature Table 1 from provides 2009 the andflow 2010. average volume Data soil for for and each pH, watering ECsystem cycle and were while system provided an by outdoor thetotal Argus weather solar station radiation. provided measures of Crop data Treatment effects on plantusing status destructive were and assessedwere completed at non-destructive on days four 5, measures. 14, stages 28on and Assessments days 41 5, after 14, seeding 25 in and 2009 33 and afteras seeding in a 2010. function Stand establishment of root-zoneemerged heating seedlings was taken calculated five from days counts aftercenter of planting of the on third 25 row cm in ofinvolved each the sub-plot. destructive Subsequent sampling assessments ofwere portions scheduled of to each either sub-plotfertility and precede treatments or (20 days followand and the 2010, 15 initiation respectively). days of Samples28 after N after collected seeding seeding on in were daysrow 2009 taken 14, (excluding from 25 25 edge cm and plants rows of were a 1 removed randomlyplastic and gently chosen bag and 7) by cooled in (5 hand each and placed sub-plot. in Whole a sealed plants from each sub-plotThereafter, ten sample representative were plants were countedwere selected removed and and from the weighed. the roots shoots withand a root razor portions blade were and weighed both collectively. shoot Theinvolved final assessment removal of30 nearly all above-ground biomass from a being processed. After weighing, separate sub-samples were dried and flash frozen for tissue elemental and biochemicalanalysis. composition Laboratory sub-samples wereharvest flash-frozen and within stored 3 at h of homogenized with 5falcon g tube (Thermo distilled, Fisher Scientific, deionizeda Pittsburgh, Kinematica water PA, 10–35 USA) in Polytron using (Kinematica, a Bohemia, NY, 50 USA). mL A the laboratory. Tissue pigment composition Anthocyanin and chlorophylldetermined after a pigment (Chlsamples extraction stored A) at from concentrations flash-frozen were tissue ) 1 1 1 O, − − − 2 stock 7H · 3 Oand Zn was 4 2 1 www.soci.org N Bumgarner − NO 2H 4 · 2011 Society of Chemical Industry 4 . All irrigation MgSO 40 cm square NH c 1 1 − 1 × − − CaSO Cu, 0.0056 g kg of nutrient solution 1 1 1 − − − ) in the fertigation solu- 1 of a micronutrient mix − in 2009 and 144 mg day 1 , 0.5 mol L consisting of reagent grade − Mo, and 0.0034 g kg 1 4 1 − 30 − SO 2 K O, 0.01 mol L B, 0.0024 g kg 2 1 total) in 2009 and 2010, respectively. 1 − − 1 2H · − 2 ) 4 total) and three times daily (10 : 00, 12 : 30, , was diluted as programmed by the Argus PO 1 4 2 ) were applied to individual root-zone heating − 1 Mn, 0.00003 g kg PO − 2 1 Ca(H S, 0.001 g kg − was automatically added to maintain pH near KCl, 0.5 mol L 1 KH 1 − N fertility (four levels) experimental units (sub-plots). 4 1 1 − − − × SO 2 The major nutrients of phosphorus (P), potassium (K), magne- Nitrogen was supplied in a separate 0.11 mol L of drip-line withemitters. At a the center measuredcapable 160–190 row of kPa, delivering these with approximately 12 36 000 a emitters mL were h total of 12 individual wileyonlinelibrary.com/jsfa in 2010, were deliveredphase to N all levels were sub-plots. decreasedeffect In of due 2010, N to treatment establishment the in concernfrom 2009 establishment that were phase due delayed fertilization. to In both residualthis experimental N runs, establishment in period sub-plots was followed21 by days (2009) a and treatment 18 period days (2010) of and where four 576 levels mg of day N (0, 72, 144 Fe, 0.006 g kg Nutrient application Fertigation was deliveredline using with 0.6 cm emittersVista, diameter at CA, soaker USA). 15 cm Each drip- spacing sub-plot contained (DIG a Irrigation 40 Products, Crop establishment Organically primed and‘Outredgeous’ pelleted lettuce but seed conventionallyME, (Johnny’s produced Selected USA) Seeds, wereApproximately Winslow, sown 1000 pre-weighed onthe seeds 32 1 sub-plots were in seven October sown rows separated byplant in 2009 7.5 density cm, each recommended an and anticipated by of the 1production. supplier for Seeds April baby were leaf 2010. lettuce evenlysubstrate and placed covered with by approximately 1 hand cm of on vermiculite. the formed (yes/no) was applied in automatic,delivered approximately programmed 1,800 mL 3-minute sub-plot cycles which at each watering.3600 Irrigation mL sub-plot was applied twice (10 : 00, 16 : 00; sium (Mg), sulfur (S), and calcium (Ca)and were sufficient provided levels at throughout consistent both experimentalthe runs. modified In Hoagland’s 2009, solution was155 mg diluted P, at 20 529 : mg(0.043, 1 K, 0.147, to 0.030, 0.081 108 deliver and mg 0.006 Mg, g kg 292 mg S, and 22 mg Ca daily hand-delivered weekly to each sub-plot. A modified Hoagland’s solution, 0.05 mol L 15 : 00; 5400 mL sub-plot to ensure the application ofnutrient the as same in total 2009. daily quantity of each 6.0. Additionally, 250 mL sub-plot system for each scheduledto watering. dilute Municipal the water was nutrientcations. used solution Fertigation and pH possiblyand provide was H additional monitored by the Argus system 0.5 mol L tion. When irrigation volume was increased inwere 2010, dilution adjusted ratios to 30 : 1 (0.029, 0.098, 0.020, 0.054, 0.004 g kg (STEM; Scotts Company0.0105 g LLC, kg Marysville, OH, USA) containing solution, diluted andthe then macronutrient solution mixedsub-plots. detailed by Prior above to the prior theplots, to Argus initiation an delivery of system N to establishment with fertilityin phase 2010 treatments of ensured in adequate 20 sub- plant daysbeginning germination N in fertility and treatments. growth 2009 During before the andsufficient establishment phase, levels 15 days of N, 288 mg day 0.5 mol L

118 119 ), 1 − trolox 1 − mol L µ ), vitamin C (g kg 1 − wileyonlinelibrary.com/jsfa ), anthocyanin, Chl A, vitamin C, 1 − ), Chl A (g kg 1 − 05 level of significance. Proc Corr was used . ), K (g kg 0 1 − < fw) levels were higher in unheated plots than P 1 − ), P (g kg BRIX) and total antioxidant power ( 1 ◦ − repetitions or runsfertility, in the timing plot ofprompted sampling establishment-phase us episodes duration and to total and analyzeaddition, run data data from duration each from of the thewithin three two destructive each sampling runs points run separately.calendar were positions In of analyzed sampling points separately andtreatments the in initiation due of each fertility to year. Moreover, theplant the total material relative quantity was of larger sampled harvests. at A Proc the Univariate third procedurenormality than was on at carried all out the data; to previousanalyzed data test further two with for using a the non-normal ProcSAS Glimmix distribution Institute, model were Cary, (SAS NC, version USA).which 9.2; This displayed skewed model distributions was that employedanalyzed following were on a more log-normal data transformation. accurately Means were back transformed for inclusion in tables. Normally distributed dataanalyzed were using Proc Mixed (1) 2009 (Chl A,(25-day N, and P, and 33-day K) biomass, and (2) vitamin 2010 and C, N N, fertilization P were and analyzed K).within as Temperature years fixed effects were and analyzed replications asGlimmix random analyses. effects in Treatment Procstatements means Mixed at and a were separated using diff to calculate Pearson correlation coefficients. RESULTS Within years, main effectsrare. Root-zone predominated heating tended and to increase biomass interactions accumulation, withholding were Nit after (Table 2), cropanthocyanin, and establishment total biomass antioxidant tended accumulation(Table power) 3). to and were Close negatively enrichment inspectiontrends reduce while related of also (e.g. highlighting interesting the exceptions. yearly data reinforces these Effects of root-zone heating on biomass Shoot fresh weight in 2009heated was significantly versus greater unheated in plots root-zone and when 41 days measured after sowing 14 (6 days,treatments days 28 and before days the 8 initiation days of and Nshoot 21 fertility fresh days weight thereafter) was (Table greater 2).unheated in plots In root-zone when measured 2010, heated 14 plots days, 25 thansowing days in and (1 33 day days after before10 the days initiation and 18 of days thereafter). N fertility treatments and Effects of root-zone heating on composition In 2009, anthocyanin (g kg sugar ( sugar or total antioxidant power levels (Table 4). Effects of nitrogen fertility on biomass In 2009, N fertilityaftertreatmentinitiation;however,shootfreshweightwaslowerin had little effect onzero-Nplotsthaninalltreatmentsafter21 shoot fresh weight daysexposuretovarying 8N days levels (Table 2). Infresh 2010, weight N measured fertility 10treatments. significantly days Biomass affected was after greatest shoot at the 576and mg initiation N, 144 of intermediate mg at N N 72 and fertility initiation, least biomass at was 0 least mg atand N. 0 576 At mg mg N 18 N plots. days and after similar N in treatment the 72, 144 equivalents g in heated plots while inN(gkg 2010 root-zone heating had no effect on Standard for 20 min 2,4,6-Tris(2- g 2011 Society of Chemical Industry 1 12,31 c × − sodium acetate 1 − ascorbate oxidase to C in a Sorvall Legend 1 ◦ − Duplicate samples of the 32 Cl, and 10 mmol L 3 m polypropylene mesh (Spectrum Fe L samples were then combined with µ Lof4UmL µ 1 µ − Each hour-long extraction took place C for 72 h and ground with a mortar and BRIX by reading on a Leica Abbe Mark II ◦ ◦ : 116–124 fresh weight (fw). A modified FRAP procedure C and then samples were centrifuged at 12,27,31 92 ◦ 1 − Samples used for vitamin C quantification were C from harvest. A 7 g frozen tissue sample was ◦ 32 2012; 80 L of water or 40 − for 15 min at approximately 20 µ -triazine (TPTZ) in a 10 : 1:1 ratio. A 6-hydroxy-2,5,7,8- s g × C. Sets of duplicate 100 ◦ Root-zone temperature and nitrogen affect the yield and secondary metabolite www.soci.org either 40 in the dark at 5 homogenized with distilled, deionized waterin as a described 1 : above 1 ratio.funnel Samples as were described then above vacuum and filtered centrifuged with at 7800 a Buchner Laboratories,RanchoDominguez,CA,USA)usingaBuchnerfunnel. Samples were thenread centrifuged on a a Beckman Coulter final DU730Coulter, spectrophotometer time (Beckman Brea, and CA,were immediately obtained USA). at 530 Anthocyanin nm and and 420 nm, Chl respectively. A absorbances Data analysis The experiment wasidentical repeated methods. in However, 2009 slight and differences 2010 between using these nearly pestle to pass a 2by mm combustion (AOAC screen. method Total 990.03). plant Fortissue other nitrogen was tissue dry was ashed elements, and analyzed acid digested and elementalwas composition determinedbyinductivelycoupledplasmaspectrometry(OSU Service Testing and Research Laboratory, Wooster, OH, USA). J Sci Food Agric 5 g sub-sample ofsequentially the homogenized with tissue 20acidified was mL, methanol. then 20 extracted mL, and then 10 mL of 1% HCl (FRASC) was also usedlettuce to quantify tissue. vitamin C levels in the frozen above described extracts were combinedsolution, with incubated for 3 precisely mL 1 h of at a room temperature,at working and read 593 nm in aFRAP Beckman working Coulter DU730 solution spectrophotometer. contained The 30 mmol L 7800 stored at degrade the vitamin C. The working solution (3was mL) detailed added above to eachfor sample and precisely incubated 1 at h.absorbances room of Samples temperature the were ascorbatefrom then oxidase the water read samples samples to were at estimate subtracted sample. the An 593 vitamin ascorbic nm acid C standard content curve and was in then each usedvitamin the to C calculate content. Frozen lettuce samples ofwere approximately 2–3 thawed g andand juiced Finishing through Inc., Ablemarle, cheesecloth NC,sucrose USA) (American sugar in content duplicate Fiber in to determine Tissue elemental composition Leaf tissue was dried at 55 refractometer (Leica Inc., Buffalo, NY, USA). buffer at 3.6 pH, 20 mmol L Tissue antioxidant power, and vitamin C andThe sugar content ferric reducing antioxidantdetermine power total (FRAP) antioxidant test power. was used to curves for cyanidin-3-glucoside (Chromadex, Irvine, CA,Chl USA) and A (Sigmacalculate tissue Aldrich, pigment concentrations St from spectrophotometric Louis,absorbances. MO, USA) were then used to pyridyl)- at 4 RT (Thermo Fisher Scientific).leaf Following tissue the and final all extraction,vacuum three filtered the extraction through volumes 297 were combined and tetramethylchromane-2-carboxylic acid (trolox)was standard used curve to convertequivalents (TE) spectrophotometric g absorbances to trolox . ) 2 − et al 0.81 0.85 0.80 − − − : 116–124 b a b a a a b a b a a a 92 0.51 0.57 0.0001 0.0001 − < < 447.6 977.9 1666.5 1294.5 2002.2 1726.1 1899.1 1990.3 2012; 0.52 0.80 NS – − − 0.76 0.85 − − J Sci Food Agric ) Final harvest (g m 2 − ) of N fertility from destructive samples 1 − b a b a c b b a 05 in Proc Mixed and Glimmix when fixed main . 0.0001 0 0.76 NS 0.81 NS < 264.9 812.7 957.5 − − < 1626.7 P 0.76 – NS 0.99 0.60 0.41 0.76 0.99 NS – 0.60 0.40 0.52 0.60 NS 0.60 – 0.51 0.51 0.41 NS 0.40 0.51 – NS − − − − 0.65 0.66 In 2010, tissue composition was similar to 2009 in that − − treatment initiation was negatively correlatedChl with anthocyanin, A, total(Table 3). antioxidant power, vitamin C andanthocyanin sugar and content totalin antioxidant tissue taken power from levelsHowever, plots a receiving were more 0 graded mg highest response Npresent in to after 2010 (Table 72, establishment. 4). 144 Unlike 2009, and vitamin C 576treatment in 2010 mg was in the N higher 0 was mg than alltreatment other was N higher treatments than and the the similar(Table 72 144 4). mg and Chl 576 A mg was higher treatment inand the 144 576 mg mg treatments, treatment which than were thelevels similar. 0, Sugar 72 were and potassium notconcentrations significantly affected measured by 18 daysfollowed N a after exposure. graded Tissue N576 response N mg treatment to N, initiation intermediate N at exposure, 72 being and greatest 144 mg at N and least at 0 mg ) Sampling two (g m 2 − 05. 0.84 NS – . 0 − = b a b a P 0.60 0.0001 − 37.5 95.1 www.soci.org N Bumgarner < 113.3 175.4 2011 Society of Chemical Industry c 0.74 0.66 NS NS – 0.57 − − 2009 2010 0.86 − ) of leaf lettuce grown with under four levels (0, 72, 144, 576 mg day 72 155.7 1286.5 72 65.3 429.9 937.8 − − + + 2 144576 133.9 144.0 1268.5 1673.5 144576 56.8 67.9 473.3 536.1 931.6 980.1 − 0.70 0.65 NS 05. NS denotes not significant at . − − 0 < P 0.70 – 0.77 0.99 0.67 0.62 0.74 0.99 0.77 – 0.63 0.62 0.86 0.77 – 0.77 0.66 0.77 NS NS NS – NS NS NS NS 0.60 0.67 0.66 0.63 – 0.45 NS 0.84 0.62 0.77 0.62 0.45 – NS − − − − − ) 0 131.4 940.0 ) 0 50.5 424.9 649.8 Biomass Antho Chl A Antioxidant Vit. C Sugar N Biomass Antho Chl A Antioxidant Vit. C Sugar N 1 1 fertility NS NS NS fertility NS NS NS − − 022) only for anthocyanin; however, root-zone . × × Heat 0.0004 0.0023 0.0067 Heat 0 Fertility NS 0.0004 Fertility NS NS 0.0003 = Heat Heat P Pearson correlation coefficients among fresh shoot biomass and tissue composition measured 41 days after sowing in 2009 and 33 days Fresh shoot weight (g m 05. NS, not significant. . 0 < Root-zone heating taken 14 days, 28 days, and 41 days after sowing inFactor 2009 and 14 days, 25 days, and 33 days after sowing in 2010 Sampling one (g m P after sowing in 2010 Biomass – Table 3. Letters denote root-zone heating and N fertility levels separated by difference statements at N Fertility (mg day 2010 Analysis of variance N Fertility (mg day 2009 Analysis of variance Table 2. effects were significant at Root-zone heating Anthocyanin Antioxidant power Chl A Vitamin C Sugar NNS wileyonlinelibrary.com/jsfa heating affected onlyfertility the effect. magnitude, Chl notsignificantly A, direction, affected vitamin of byTissue C the N N concentrations N and 21 fertility days sugar after ashighest N and content treatment a similar initiation at were were main 576than and not effect 144 the mg 576 N (Table mg 4). with 0 plots.at and 576, Phosphorus 72 144 concentrations mg lower and weregroup. 72 similar Potassium mg levels N at but 0 and higherthan 576 than mg at N in were 72 the similar and but 0 lower mg 144 N mg control N (Table 4). Biomass at 21 days after N Effects of nitrogen fertility on composition In 2009, anthocyanin and totaltissue antioxidant power taken was from highest in plots0 receiving and zero 72 N,576 mg mg while N plots plots recorded with receiving The statistical statistically intermediate interaction values higher between root-zone atsignificant values heating ( 144 and than mg N was N the (Table 4).

120 121 In ) 1 − b a a b Overall, 38–40 45.0 39.5 64.4 72.1 68.9 61.9 42.3 45.5 35 )K(gkg 1 − a b a a a b a a 6.9 7.5 7.5 8.6 9.0 9.0 5.9 7.1 which can be a human )P(gkg 1 − wileyonlinelibrary.com/jsfa 36,37 a c b ab a c b b solution. Supplies of non-target 56.956.9 7.8 9.2 62.4 71.2 3 NO 4 a b BRIX) N (g kg 2.12.1 46.8 58.7 3.8 2.4 2.42.2 31.9 44.4 ◦ Sugar ( 05 in Proc Mixed and Glimmix when fixed main . tends to enhance crop growth and reduce can be temperature-limited as well. 0 − − < 3 3 c a b a b c fw) P 1 − 0.0001 NS 0.0002 0.022 NS 0.066 0.071 0.074 0.034 0.0570.0430.047 3.20.055 2.8 2.9 46.8 3.1 56.0 60.2 64.7 0.158 0.122 < and NO Vitamin C and NO (g kg + + 4 4 fw) Nitrogen (N) was supplied separately from the other essential 1 − to provide a solid,the inorganic root medium system but that lacked physicallyof the supported nitrogen organic mineralization potential substrates.provide cation Calcinated exchange clayimprove vegetable and was production buffering in also inorganic capacity mediums. included reported to to nitrate accumulation in leafy crops, nutrients remained consistent among allas treatments. NH N supplied health hazard. Also,equalized the the opportunity for presence plant N of uptakeammonium across both seasons and since N nitrate formsand uptake high may appear soil have to temperatures,of respectively, be NH although preferred translocation at low follow-up work, this system wouldform to allow be N set according delivery to amount root-zone and temperature. Effect of root-zone heating on biomass Overall,trendsinleaflettucebiomassandcompositionweresimilar across years. However, ambientwhether conditions temperature appeared or N toaccumulation levels dictate and composition were in any most one year. limiting Conditions inwere to 2009 less biomass conducive to growth thanheating in 2010; effects as on a result, biomass root-zone 2009 and relative to composition 2010. were In 2010, enhancedwere however, diminished root-zone in and heating N effects treatment effects were enhanced. the outdoor experimental systemwith integrated the impedance a of rooting soil with medium typical temperature of and indoor nutrient soil-less control systems. plant nutrients in an NH trolox 1 c a b a b bc c a b c − 05. . 0.0001 NS NS 0.0020.0001 0.011 0.0052 0 16.6 15.3 19.8 13.7 27.9 19.4 < < = mol L µ P ( Antioxidant power equivalents g 2011 Society of Chemical Industry b b b a a b fw) c 1 − 0.0001 0.0021 0.0084 0.0015 NS NS NS 0.481 0.471 0.479 0.518 0.663 0.530 0.6170.5910.5830.595 22.4 16.2 14.7 13.8 < (g kg Chlorophyll A fw) a b c c a b a b bc c 1 − 0.469 0.0001 NS 0.00010.403 0.032 0.511 19.8 0.114 2.3 46.3 6.8 43.4 0.305 0.373 0.464 18.8 0.094 2.1 44.6 6.9 42.8 < < 05. NS denotes not significant at (g kg . Anthocyanin 0 < 72 0.386 72 0.364 P − − + + 576 0.300 144 0.320 144576 0.328 0.316 Here, sand was mixed with calcinated clay : 116–124 ) 0 0.610 ) 0 0.543 1 1 34 92 − − fertility 0.022 NS NS NS NS NS NS NS fertility NS NS NS NS NS NS NS NS 2012; × × Heat NS NS NS NS NS NS NS NS Heat 0.0003 Factor Tissue composition of leaf lettuce grown under varying root-zone heating and nitrogen fertility treatments 41 days after sowing in 2009 Fertility Fertility Rootmorphology,inturn,caninfluencenutrientuptake 33 Heat Heat Root morphology and architecture can vary with rooting N Fertility (mg day Letters denote root-zone heating and N fertility levels separated by difference statements at 2010 Analysis of variance 2009 Analysis of variance Table 4. and 33 days after sowing in 2010 effects were significant at Root-zone heating Root-zone heating N Fertility (mg day Root-zone temperature and nitrogen affect the yield and secondary metabolite www.soci.org J Sci Food Agric medium (e.g. liquid versus solid), primarily due toimposes. the impedance it DISCUSSION Growing system and nitrogen supply The primary goal ofperature this work and was nitrogen toaccumulation document fertilization and root-zone composition. effects tem- These onin effects highly lettuce have controlled been biomass indoor tested resembling field settings conditions. but The specialized rarely andture costly in common infrastruc- to environments highly controlled systems isproduction prohibitive settings. in And, typical root andliquidversussolidrootingmedia-basedculturesystems. shoot parameters can Therefore, varyour in secondary goal wastreatments to using impose a the cultural temperature systemimental and offering control fertility but high with levels physical ofsoil, properties exper- thereby more promoting similar root to growth field thatfield may conditions. be The expected system under used hereuse is in scalable experimental and and suitable limited for commercialcomponents settings include and electric its heating main cables (replaceablewater) with and an hot inorganic, chemically stable and potentially reusable rooting medium. Use of the Argusalso Titan served Nutrient our specific Delivery experimental System objectives. N. As a group, tissue144 and P 576 concentrations mg following N were exposureN. similar to Final but 72, yield greater than was in positivelywith plots and elemental given leaf significantly no N correlated and negatively (Table correlatedvitamin 3) with C, anthocyanin, sugar, and total antioxidant power. and other variables. . , a 53 et al Pythium Pythium : 116–124 92 2012; variation in these 52 Lower N utilization dur- 47 ) at final harvest tended to 1 and this may be due to − vitamin C, anthocyanin, and In the presence of 51 J Sci Food Agric 50 spp. was identified. Banrot 40WP (Scotts 14–17,24,55–57 however, data here suggest that temperature and Pythium 22,54 In 2010, the establishment period N fertility level was reduced Like for biomass yield, N supply effects on indices relevant Nitrogen fertilization effects on plant biomass present interest- Unlike N, P and other measures of tissue composition, the and warmer ambientconditions sped temperatures the onset prevailed; of cleardifferences together, N treatment in effects. these Significant cropafter biomass sowing were (10 evident daysrespectively). 25 and days Interestingly, 18 and differences days 33 infour after days N biomass fertility N treatments between were treatment greater the In at initiation, the 25 days final than week at of 33observed the days. and 2010 experimental run, plantCompany wilting LLC) was was appliedis as a a common drench root at pathogenless a capable lettuce of label production reducing rate. growth systems. in soil- reduced plant growth orlevels mortality of have fertility been linked in to some higher crops, light may affect Chlconditions. A levels more strongly than N underto some human nutrition wereon greatest in these 2010. aspects Yet,not annual of N direction. tissue effects secondary As composition metabolites, varied in in other magnitude, reports on the impact of N on ing early growth stages and low temperature periodsby can tailored be offset fertigation regimes. Regardless,here yield suggest maximum values production is reported possible underthe reduced spring N in and fall in temperate climates. Effects of nitrogen fertility on composition Elemental composition levels followed similar patterns in 2009 and 2010. Plant tissue N and P levels (g kg increase with N application intracked both years. N Tissue N treatment concentration P more closely levels, than althoughdiffer P greater significantly among concentrations the than three since N indeficiency treatment groups. has the Nitrogen prompted variable control responses in group, P did and K not levels, total antioxidant power levelsremoved. Moreover, tended anthocyanin, vitamin to C and increasecapacity total when antioxidant values N were was and consistently 2010 positively (Table correlated 3),power in but levels 2009 only were anthocyanin significantly and greater total at antioxidant low N levels in both calculated estimates of Nreduce N usage losses by more reinforces closely matching theplant N growth fertilization need and rates temperature with to patterns. further increased susceptibility to root disease.temperatures The and presence root of pathogens warmer soluble coupled salts with in higher the levels mediumlevel of as may measured have 33 limited days after the sowing. response to N ing implications for earlyutilization and calculations revealed late that season plantsroot exposed production. zones to but Nitrogen unheated high fertilityof incorporated the N lowest applied percentages whileheated incorporation root zones rates and were at low generallyincorporation to higher intermediate rates N in were levels. Calculated 14–48%While both in ranges 2010 overlapcovery but previous in lettuce 5–23% estimates produced of outdoors in on 12–23% sand, 2009. N re- result noted here. response of Chl A tosignificantly N affected treatment differed by by N year.was Chl true treatment A in in was 2010 not 2009 whenN Chl but treatment A the levels level. opposite wereavailability; Chlorophyll greatest at levels the are highest thought to follow N 47,48 Cin ◦ 11,12,43 Still, the www.soci.org N Bumgarner 49 2011 Society of Chemical Industry c C warmer in 2010 than ◦ Light levels also could have 46 However, the effect was greatest in C in 2010. Temperatures in fall 2009 12,29,40,44,45 ◦ 36,41,42 warmer than historical values. Results here suggest that rising For leaf biomass specifically, the presence of root-zone heating ◦ increased shoot fresh weight in both years, anwith outcome previous consistent reports. 2009 and corresponded togiven the ambient temperatures. potential Root-zone impact heatingweightrelativetounheatedplotsby270%in2009and55%in2010. increased of shoot soil heating Average air temperatures were nearly 5 wileyonlinelibrary.com/jsfa an assertion consistent with the law of the minimum. Effects of nitrogen fertility on biomass Biomass accumulation amongshowed the similar trends foursamplings in N taken 2009 28 fertility and and 4133 treatments days 2010. days after after However, sowing sowing data (2009) (2010)varied and suggest from across 25 that years. and shoot Higherand growth N patterns lower levels during growing standperiod temperatures establishment typified throughout 2009. the Thesetime entire conditions necessary may study have forbiomass increased levels fertility varied the with N effects treatmenttreatment at to 41 initiation) days but become (21 not days at after apparentinitiation). N 28 Shoot days biomass since measures, (8 therefore, days varied after littletreatments among N treatment for theby majority its of conclusion,significantly the biomass from values experimental the for period. zero-Nprevious all control. And, reports N These suggesting findings treatments agreewhen that differed other with environmental fertility factors, such impacts as temperature,limiting can are more to diminish plant growth and productivity (e.g. as in 2009), Phenolic, anthocyanin, vitamin C, sugarand and other Chl levels crops inshoot-zone lettuce temperatures. have risen in response to reduced root or resembled long-term values providedData by Center the (http://www.ncdc.noaa.gov/oa/mpp/freedata.html); Nationalhowever, Climatic temperatures in spring5 2010 averaged approximately contributed to the oftenversus 2010 higher as greater levels pigment concentrations offor may have maximal Chl allowed levels recorded of light in interceptionthe and 2009 lower photosynthesis light under conditions of 2009. Effects of root-zone heating on composition Root-zone temperature alsoAnthocyanin, affected Chl leafantioxidant A, tissue power vitamin composition. were higherin C in 2009. and unheated However, thanheatingdidnotinfluencetheseleaftissuecompositionparameters. sugar heated in plots levels, theThese warmer and results season total arethe of consistent 2010, influence with root-zone metabolism of previous and findings low the outlining and levels of high anthocyanins temperature specifically. on secondary spring-time temperatures facilitated the onset andN progression treatment of effects more so than declining fall-timeHowever, temperatures. this hypothesis requires further testingpossibly in with field additional settings, variables such as light. presence of N-fixing microbial associationsgrowing on medium the surface may ofproviding the have 72, also 144 contributed oryield in 576 to 2009 mg (B. McSpadden-Gardener, the N personal communication). had fact little that influence on biomass in 2009 and,of therefore, root-zone generally heating. supportedhave Likewise, growth a root-zone regardless more heating pronounced effectthan appeared in on 2010 to temperature since the profiles average in sub-surfaceplots temperature 2009 of exceeded heated that of unheated plots by more than 8 2009 but by less than 6

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