Selection During Crop Diversification Involves Correlated Evolution of The

Research

Selection during crop diversiﬁcation involves correlated evolution of the circadian clock and ecophysiological traits in Brassica rapa

Yulia Yarkhunova1, Christine E. Edwards2, Brent E. Ewers1, Robert L. Baker1, Timothy Llewellyn Aston1, C. Robertson McClung3, Ping Lou3 and Cynthia Weinig1,4 1Department of Botany and Program in Ecology, University of Wyoming, Laramie, WY 82071, USA; 2Center for Conservation and Sustainable Development, Missouri Botanical Garden, St Louis, MO 63166, USA; 3Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA; 4Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA

Summary Author for correspondence: Crop selection often leads to dramatic morphological diversification, in which allocation to Cynthia Weinig the harvestable component increases. Shifts in allocation are predicted to impact (as well as Tel: +1 307 7666378 rely on) physiological traits; yet, little is known about the evolution of gas exchange and Email: [email protected] related anatomical features during crop diversification. Received: 26 June 2015 In Brassica rapa, we tested for physiological differentiation among three crop morphotypes Accepted: 14 October 2015 (leaf, turnip, and oilseed) and for correlated evolution of circadian, gas exchange, and phenological traits. We also examined internal and surficial leaf anatomical features and biochemical New Phytologist (2015) limits to photosynthesis. doi: 10.1111/nph.13758 Crop types differed in gas exchange; oilseed varieties had higher net carbon assimilation and stomatal conductance relative to vegetable types. Phylogenetically independent contrasts Key words: Brassica rapa, circadian rhythms, indicated correlated evolution between circadian traits and both gas exchange and biomass crop diversification, ecophysiological traits, accumulation; shifts to shorter circadian period (closer to 24 h) between phylogenetic nodes Farquhar model, leaf anatomy. are associated with higher stomatal conductance, lower photosynthetic rate (when CO2 supply is factored out), and lower biomass accumulation. Crop type differences in gas exchange are also associated with stomatal density, epidermal thickness, numbers of palisade layers, and biochemical limits to photosynthesis. Brassica crop diversification involves correlated evolution of circadian and physiological traits, which is potentially relevant to understanding mechanistic targets for crop improvement.

Introduction Selection during crop diversification may lead to correlated evolution of multiple traits. Evolution of some traits (e.g. mor- In his early study, ‘The variation of plants and animals under phological ones) may be functionally dependent on other traits domestication’, Darwin recognized that studying domesticated (e.g. physiological ones). During domestication or diversification, plants provided insights into fundamental evolutionary processes improvements in size or in relative allocation to the harvestable (Darwin, 1868). In the process of domestication, a crop species component are probably enabled by physiological evolution. For becomes evolutionarily differentiated from a wild progenitor as instance, sink strength appears to be greater in oilseed vs veg- humans transition from harvesting and stewardship of plants in etable crops within a species, because oil per unit mass is more wild populations to imposing selection on plants in an agricul- metabolically expensive to produce than vegetative organs; car- tural ecosystem. This is commonly distinguished from crop bon assimilation is correspondingly higher in oilseed varieties diversification, which occurs after domestication as humans select (Evans, 1996; Ruuska et al., 2004; Goffman et al., 2005; Li et al., for secondary traits such as crop growth in diverse geographic 2006). A small number of recent metabolomic studies also sug- regions, synchronized timing of life-history events, or enhanced gest correlated morphophysiological evolution (Del Carpio et al., flavor (Meyer & Purugganan, 2013; Abbo et al., 2014). Among 2014; Hennig et al., 2014). Nevertheless, despite the clear mech- the most visually apparent phenotypic changes during crop diver- anistic links in contemporary populations and across species sification, dramatic morphological differences have emerged (Wright et al., 2004; Sack et al., 2013) and the potential relevance among related vegetable crops (Tsunoda et al., 1980; Purugganan to crop improvement, examples of joint morphophysiological et al., 2000) and between related vegetable and oilseed varieties evolution during crop domestication or diversification are com- (De Vries, 1997) as a consequence of selection to increase the paratively rare (Cook & Evans, 1983; Smartt, 1988). Simultane- harvestable component. ously quantifying multiple morphological, physiological, and

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developmental traits in domesticated crops that have undergone explored if crop types differ in physiological (gas exchange) traits subsequent diversification should offer insights as to suites of and, if so, whether gas exchange covaries phylogenetically with morphophysiological traits that evolve jointly to enhance yield circadian period. Further, we tested the related gas exchange (Evans, 1996). hypothesis in the crops: the rate of photosynthetic carbon Many developmental features could contribute to physiologi- assimilation (A) is limited by gas supply of CO2 (gs) and bio- cal diversification among crop types. One well-known regulatory chemical demand for CO2; demand is, in turn, regulated by control on physiological traits is the circadian clock. Circadian light-dependent and light-independent processes occurring in the rhythms are biological oscillations that are c. 24 h in duration mesophyll, which are either limited by light harvesting (ribulose- and coordinate the diurnal timing of many physiological and 1,5-bisphosphate (RuBP) regeneration) or by sugar metabolism metabolic processes in plants. The circadian clock regulates eco- through CO2 assimilation (Vcmax, maximum carboxylation physiological traits (e.g. starch utilization and photosynthesis) in capacity of Rubisco) and triose phosphate utilization. Finally, we experimental genetic lines such as mutant genotypes that express examine leaf anatomical features (stomatal density and internal discrete clock phenotypes that differ from the cognate wild-type leaf features) that may contribute to physiological diversification. (Dodd et al., 2005; Graf et al., 2010; Faure et al., 2012). Quanti- We explore how stomatal density and thickness of epidermis tative clock variation is also associated with net carbon assimila- might contribute to CO2 supply and resistance differences, and tion and stomatal conductance in segregating progenies of how palisade structure could affect light absorption, including Brassica rapa (Edwards et al., 2009, 2011; Lou et al., 2011), electron transport and carbon fixation. potentially because clock cycles closer to 24 h enhance diurnal timing of gas exchange. During crop diversification, the circadian Materials and Methods clock and gas exchange traits may evolve in a correlated manner if gas exchange responds to allele frequency changes at clock loci Study system or if photosynthetic evolution alters sugar status and thereby affects clock function (Dalchau et al., 2011; Moghaddam & Van Brassica rapa L. (Brassicaceae) is an internationally important den Ende, 2013). More proximately (and quite possibly indepen- crop, and distinct subspecies are cultivated as oilseed crops (ssp. dent of the circadian clock), leaf anatomical features or photosyn- oleifera), leaf vegetable crops (ssp. chinenesis and pekinensis), and thetic biochemistry could contribute to crop variation in gas root vegetable crops (subsp. rapa). Accessions used here reflect exchange (Kebede et al., 1994). the global cultivation of the species; accession origin and Crucifers provide some of the most dramatic examples of germplasm identifiers are presented in Supporting Information crop morphological diversification. Reproductive organs are Table S1. highly altered in some members of Brassica oleracea, such as cauliflower and broccoli (Purugganan & Fuller, 2009). Vege- Circadian measures tative features are also highly modified among varieties of both B. oleracea and B. rapa. Distinct B. rapa accessions have In all, 41 accessions were screened for circadian period undergone selection for increased allocation to above-ground (Table S1). Some accessions were screened previously by Lou organs (i.e. leaves in cabbage, pak choi, and turnip green et al. (2011). Here, previously published data, data for additional crops and seeds in oilseed types) as well as below-ground replicates of accessions within the original panel, and data on organs (i.e. enlarged root-like hypocotyl in turnips). Accompa- additional accessions are analyzed to test for circadian differences nying morphological evolution, we hypothesize that crop types among accessions that generalize to crop type; within each acces- will exhibit physiological diversification as a consequence of sion, between eight and 15 replicates were screened. For details differences in overall size, sink strength, and proportionate of the circadian screens, which used leaf movement as a proxy to allocation; the strength of morphophysiological correlations estimate circadian period, see Lou et al. (2011) as well as reviews remains as an empirical gap in our understanding of evolu- of this method (McClung, 2006; Greenham et al., 2015). tion during domestication and diversification, and may inform targets for future crop improvement. Further, past studies Leaf gas exchange measurements provide evidence of genotypic variation in circadian clock period (Lou et al., 2011), but it remains unclear if circadian In addition to differentiation of the circadian clock, we were differences generalize to crop morphotype or if circadian phe- interested in whether crop types differ in gas exchange, and notypes correlate with physiological traits over evolutionary whether there is evidence of correlated evolution between circa- time, which may again inform breeding for crop improve- dian and gas exchange traits. A subset of crop accessions ment. (Table S2) was sampled for physiological and leaf anatomical Our overall aim was to test if the morphological variation aris- traits. For gas exchange experiments, nine to 10 replicates of 21 ing from selection for different crop types is associated with dif- representative genotypes were grown from seed in growth cham- ferences in circadian, leaf gas exchange, and anatomical traits in bers (PGC-9/2 with Percival Advanced Intellus Environmental different B. rapa accessions. With this study we accomplished the Controller, Percival Scientific, Perry, IN, USA). Seeds were following specific aims: we first tested whether genotypic differ- planted into 6 9 6 9 9 cm plastic pots filled with Redi-Earth pot- ences in circadian period generalize to crop type; we then ting mix (Sunshine Redi-Earth Professional Growing Mix, Sun

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Gro Horticulture, Bellevue, WA, USA) and including a slow- Leaf morphological measures release fertilizer (Scotts brand Osmacote Controlled Release Clas- sic, NPK; Scotts, Marysville, OH, USA). Three seeds were Stomatal density was measured in a subset of plants on which gas planted in each pot, and then covered with vermiculite and satu- exchange data were collected. The fourth leaf was chosen for rated with water. Plants were placed in the growth chamber with stomatal measurements; this leaf was fully expanded and was following conditions: photoperiod of 15 : 9 h, 24 : 22°C, developmentally close to the third leaf, which was used for gas light : dark cycle, and photosynthetic photon flux density = exchange measurements. Each leaf was first preserved in forma- À À 500 lmol photons m 2 s 1. Plants were thinned to one seedling lin-aceto-alcohol (FAA) solution (ratios of ethanol, ethanoic acid, per pot after cotyledons had expanded (c. after 5 d). At subjective and formalin were, by volume, 18 : 1 : 1), following several dawn, plants were watered daily to retain consistently high soil ethanol washes over the course of 1 wk until the leaf tissue moisture. became transparent. An aqueous solution of toluidine blue O Gas exchange measurements were taken using a Li-Cor LI- (0.05%) was used to stain the tissue for 30–60 s depending on 6400 XT portable infrared gas analyzer with leaf chamber fluo- the thickness of the leaves. Light micrographs were taken using rometer (Open System Vers. 4.0, Li-Cor Inc., Lincoln, NE, an Olympus BH2 compound light microscope at a magnification 9 USA). Measured traits were maximum photosynthetic rate (Amax) of 500. Within each leaf sample, stomata were counted visually and stomata conductance (gs). Measurements were taken from in five to six areas chosen randomly. Five to six squares of dimen- the third fully developed true leaf at least 1 h after subjective sions 200 9 200 lM were used to count stomata visually. The dawn under the following chamber conditions: photosynthetic range number of stomata per square was 10–35. The software = l À2 À1 = photon flux density (PPFD) 1500 mol m s , ref [CO2] program, Image J (Schneider et al., 2012), was used to calculate l À2 À1 = ° 400 mol m s , Tleaf 24 C, and vapor pressure deficit based stomatal density, that is, the number of stomata counted within on leaf temperature (VPDL, kPa) was kept between 1.3 and an image area. In total, measurements were taken from five to six 1.7 kPa (Long & Bernacchi, 2003). Measurements were taken images within each of six to eight replicates in each of 15 geno- 3–5 min after all chamber conditions stabilized. types (equally representing the three crop types) (n = 639).

A–Ci response curves Leaf anatomical measures – A Ci response curves were used to estimate biochemical limits Tissue samples were fixed in FAA and stored in 70% ethanol. to photosynthesis, including the maximum rate of RuBP car- Subsequently, leaves were dehydrated in a standard ethanol gradi- boxylation (Vcmax) and the maximum rate of electron transport ent. Ethanol was replaced with Histoclear, and the tissue was ° driving RuBP regeneration (Jmax). In further regard to Jmax, slowly infiltrated with paraffin at 60 C (Fowke & Rennie, 1995). RuBP regeneration is controlled by the amount and activity of Samples were embedded in paraffin, serially sectioned at 10 lM, Calvin cycle enzymes as well as NADP and ATP pool sizes, and stained with toluidine blue O (Sakai, 1973). Light micro- while NADP and ATP are supplied by the light-dependent graphs were captured using a Zeiss Primovert compound light reactions and are thus limited by electron transport between microscope and Zeiss camera and AxioVision software the two photosystems. (AxioVision Rel 4.9.1; Carl Zeiss Imaging Solutions). The transi- Response curves were performed on the same leaf as the single tion from spongy mesophyll to palisade parenchyma was delin- time point measurement again using six Li-Cor 6400 XT eated by drawing a straight line through the center of multiple portable photosynthesis systems within a 5 d period for the vascular bundles. All measurements were collected from a growth chamber experiment. Because of the time-consuming 1000 lM long area of tissue in Photoshop CS4. Internal leaf – nature of A Ci phenotyping (in which measurement of one repli- anatomy was likewise measured in a subset of samples including cate can take 1–2 h and measurements can only be taken during two to five replicates in each of nine genotypes (equally represent- an approx. 2 h time window daily) and the fact that all replicates ing the three crop types) (n = 24), owing to the extensive time – should be measured at a similar, preflowering stage, A Ci curves needed to clear, fix, embed, section, and analyze histological fea- were collected only on three to four replicates within each of four tures. genotypes for each of the three crop types (n = 44). Chamber À À conditions were maintained at PPFD = 1500 lmol m 2 s 1, = ° Plant harvest, leaf area, nitrogen content Tleaf 25 C and VPDL between 1.3 and 1.7 kPa. Reference CO2 concentration in the chamber was controlled with a CO2 After 5 wk of growth, plants were carefully removed from pots, mixer across the series of 400, 500, 600, 800, 1000, 1250, 1500, and roots and shoots were washed to remove soil. One leaf per À À 2000, 400, 300, 200, 100 and 50 lmol m 2 s 1 and mea- plant was scanned using the Epson Perfection V500 software at a surements were recorded after equilibration to a steady state resolution of 150 dpi (Epson, Nagano, Japan). The software pro- – (5 7 min). The utility tool A/Ci Response Curve Fitting 10.0 gram IMAGEJ was used to calculate leaf area (Schneider et al., available at http://landflux.org/Tools.php, which is based on the 2012). Plants were cut at soil level, and the single collected leaf, equations of Farquhar et al. (1980) and an approach developed the remaining above-ground portion, and the below-ground por- ° by Ethier & Livingston (2004), was used to estimate Vcmax and tion were dried at 65 C for 72 h. DW was then measured. The – d15 d13 Jmax for each A Ci curve. oven-dried leaves were ground and analyzed for N, C, leaf

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nitrogen concentration (wt% N), leaf carbon concentration (wt% analyses (1000 replicates) (Felsenstein, 1985) were used to assess C) and C : N ratio using an elemental analyzer (ECS 4010, branch support employing a heuristic search with TBR branch Costech Analytical Technologies Inc., Valencia, CA, USA) cou- swapping, with 10 random additions per replicate, saving no pled to a continuous-flow inlet isotope ratio mass spectrometer more than 100 trees per replicate. (CF-IRMS; Delta-plus XP, Thermo Scientific, Waltham, MA, To calculate correlated changes in traits using a phylogenetic USA). N data were collected on seven to eight replicates within framework, we calculated PICs based on the most parsimonious each of 12 genotypes equally representing the three crop types. trees resulting from phylogeny reconstruction. The PIC approach is a comparative one that assumes a Brownian motion model of evolution to test for evolutionary correlations of traits after Data analysis explicitly accounting for phylogenetic relatedness. All PICs were Statistical approach and data treatments We used ANOVA carried out using the PDAP module in MESQUITE (Midford et al., with post hoc Tukey tests to identify significant crop type and 2005). The two most parsimonious trees with stored branch genotype within crop type effects (PROC GLM, SAS v.9.4., SAS lengths (see the Results section) were used to compute contrasts. Institute Inc., Cary, NC, USA). We observed a small number of Before carrying out analyses, we checked diagnostic plots of the outlier genotypes within crop types for circadian period. Notably, absolute values of the standardized PICs vs their standard devia- our assignment of ‘crop type’ and history of selection was based tions using the most parsimonious trees to determine whether the on germplasm annotation, but our observation of morphology branch lengths of the phylogenetic tree adequately fitted the tip and phenology suggested that some genotypic outliers might have data (Garland et al., 1992; Diaz-Uriarte & Garland, 1996, been better assigned to a different crop type. For instance, in the 1998); because branch lengths were adequately standardized for group of oilseeds, the genotype WO-024 had an enlarged below- all characters on both trees, these analyses are not discussed fur- ground storage structure by midseason reminiscent of a turnip, ther. We used PICs to carry out two different types of analyses. and had a period phenotype that resembled other vegetable rather First, we calculated an independent contrast between circadian than oilseed types (Fig. S1). Likewise, several turnips had reduced period and each gas exchange and allocation trait as well as flow- vernalization requirements similar to oilseeds, and showed period ering time, all of which are continuous traits, to understand pat- lengths closer to the average oilseed genotype. We therefore terns of correlated evolution between these traits. Second, we tested for genotype (crop type) and crop type effects either using used PICs to assess whether shifts in crop type (which is a cate- the germplasm designation to assign genotypes to a crop type or gorical trait) were associated with significant shifts in gas using phenotypic similarity to assign genotypes to crop type. exchange and allocation traits following Whittall & Hodges Results were not affected by these differences in assignment, and (2007). We calculated contrasts and then used a paired t-test to we therefore present the more conservative results using the calculate whether contrasts resulting from a shift in crop type germplasm designation (Table S1). were significantly different from contrasts between nodes of the We used linear regression to test for associations between same crop type. We also repeated these analyses with all most- quantitative clock variation and gas exchange traits, biomass parsimonious trees resulting from phylogeny reconstruction and accumulation, and flowering time. We used both unadjusted val- using trees that varied in rooting to assess how tree topology and ues of gas exchange in these analyses as well as Amax values rooting affected the results of the PICs. Because all results were adjusted for CO2 supply limitation and gs values adjusted for bio- consistent regardless of variation in topology and rooting, these chemical feedbacks on stomatal behavior. More specifically, we analyses are not discussed further. performed the genotypic regression of Amax =gs to generate resid- uals and the variable rA ; rA thus reflects variation in carbon max max Results assimilation after CO2 supply limitation is considered. The variable rgs reflects variation in stomatal behavior after biochemical Using ANOVA, we found significant effects of genotype (crop demand is considered. type) on circadian period (Tables S1, S3a). We further found a significant effect of crop type on circadian period duration Phylogenetic analysis and phylogenetically independent con- (Fig. 1; Table S3a). In particular, we observed that oilseeds have trasts To reconstruct a phylogeny, we used amplified fragment circadian cycles c. 1 h shorter on average and closer to 24 h than length polymorphism data from a previously published study those of leaf crops or turnips (Fig. 1). (Zhao et al., 2005), which consisted of 527 aligned characters Analysis of gas exchange data showed that crop types also differ from 21 accessions representing three crop types (seven oilseeds, significantly in both maximum net photosynthesis rate (Amax) six leaf crops, and eight turnips; Table S1). Phylogeny recon- and stomatal conductance (gs), such that oilseeds have higher val- struction was carried out using a parsimony optimality criterion ues of both traits relative to leaf crops and turnips (Fig. 2a,b; in PAUP* 4.0b10 (Swofford, 2002). In all parsimony analyses, Table S3b,c). A strong positive association was observed between heuristic searches were conducted using 1000 random addition Amax and gs (Fig. 3a). No significant correlations were observed replicates and tree bisection and reconnection (TBR) branch between unadjusted gas exchange values and circadian period swapping, saving 100 trees per replicate. All most parsimonious lengths (data not shown). However, rAmax (which accounts for trees were stored, saving branch lengths, which were used for genotypic variation in stomatal conductance and CO2 supply) phylogenetically independent contrasts (PICs). Bootstrap and circadian period were positively correlated (Fig. 3b). rgs

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(b) Fig. 1 Circadian period length among different crop types estimated by cotyledon movement. Error bars indicate Æ 1SE. Different letters indicate statistically signiﬁcant differences among Brassica rapa crop types (P < 0.05).

(which accounts for biochemical feedbacks on stomatal behavior) and circadian period were negatively correlated (Fig. 3c). Thus, circadian period is related to leaf gas exchange once gas supply or demand is statistically considered. Biomass was positively associated with circadian period (Fig. 3d), suggesting that the high rAmax of the leaf crops and turnips (genotypes with long circadian period; Fig. 3b) is important to determining mass or simply that their preferential allocation of fixed carbon to vegetative organs rather than to metabolically expensive seed oil affects total biomass accumulation. No association was observed between circadian period and the phenological trait, flowering time (data Fig. 2 Differences in gas exchange traits among crop types. (a, b) Stomatal conductance (a) and maximum rate of CO2 assimilation (b) in Brassica not shown). À À rapa crop accessions measured at 1500 lmol quanta m 2 s 1 and a leaf The significant association between circadian period and both temperature of 24°C. Error bars indicate Æ 1SE. Different letters indicate gas exchange parameters and biomass (Fig. 3) can be explained statistically significant differences among crop types (P < 0.05). either by inheritance of a combination of traits from a common ancestor (e.g. an oilseed ancestor that had both high gas exchange values and short 24 h circadian cycles) or by selection for corre- that there is a limited number of discrete transitions between lated evolution of these two traits. Using PICs to assess correlated nodes (crops) on the phylogeny and therefore low power to detect evolution while taking phylogenetic relationships into account, traits associated with these transitions. we observed significant correlations between circadian period and We were interested to further explore the source of genotypic = = = = rAmax (r 0.53, P 0.02) and total biomass (r 0.47, P 0.05) differences in gas exchange parameters, Amax and gs. In order to and a marginally significant correlation between period and rgs explain limitations on photosynthetic rates, we measured the (r = À0.45, P = 0.06), as illustrated in Fig. 3. These results sug- response of photosynthesis to changes in intercellular mole frac- – gest that observed correlations are significant not because phylo- tions of CO2 (A Ci curves) in a subset of genotypes using the genetic groups have a common inherited relationship between Farquhar–von Caemmerer–Berry biochemical model of photo- – these traits, but because evolution in circadian rhythms is accom- synthesis. Fig. 4(a) shows the A Ci curve in three crop types of panied by a corresponding response in the other traits regardless B. rapa and two parameters important to photosynthetic perfor- of phylogenetic relationships. In the second analysis, we used mance, including the initial slope (which denotes the carboxyla- PICs to assess whether shifts in crop type (which is a categorical tion-limited region where CO2 supply limits the activity of trait) between phylogenetic nodes were associated with shifts in Rubisco while RuBP is not limiting) as well as the plateau (which gas exchange and allocation traits, but we did not find any signifi- is the RuBP-limited region where CO2 no longer limits the car- cant association between crop diversification and evolution of boxylation reaction, but availability of RuBP limits the activity of these traits. This may reflect an absence of trait evolution Rubisco). Carboxylation-limited and RuBP-limited regions attributable to independent crop diversification events, or simply appear qualitatively similar in turnips and leaf crops, while

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Fig. 3 Association among circadian period, gas exchange traits, and biomass. Each symbol represents a genotype; crop types are coded by color. (a) Association between

maximum net photosynthesis rate (Amax) and stomatal conductance (gs) for 21 genotypes of three Brassica rapa crop types. The line represents the following relationship: r = 0.79 (R2 = 0.61), P < 0.0001. (b) Association between maximum net photosynthesis rate (c) (d) (Amax) and circadian period after statistically factoring out stomatal conductance (gs). The line represents the following relationship: r = 0.63 (R2 = 0.36), P = 0.0038. (c) Association between stomatal conductance

(gs) and circadian period after statistically factoring out Amax. The line represents the following relationship: r = À0.58 (R2 = 0.29), P = 0.0091. (d) Association between circadian period and total biomass accumulation. The line represents the following relationship: r = 0.56 (R2 = 0.27), P = 0.0117.

oilseed replicates appear to differ from the vegetable crop geno- harvesting, and photosynthethic biochemistry. No crop type dif- types in both features. Analysis of the curves indicates that Vcmax, ferences in abaxial epidermis were observed. the maximum Rubisco carboxylation rate, is in fact significantly higher in oilseeds relative to leaf crops and tends to be higher Discussion in oilseeds relative to turnips (Fig. 4b; Table S3d). The values of Jmax, the maximum electron transport rate driving RuBP In the present study, we examined hypotheses related to corre- 2 = regeneration, and Vcmax are significantly correlated (R 0.55, lated morphophysiological evolution. To test for correlated trait P < 0.001). Most of the points fall close to a straight line, and the evolution, we measured circadian rhythms, gas exchange, photo- slope of the fitted regression line equals 1.33, indicating that an synthetic biochemistry proxies, and leaf morphological and increase in intracellular CO2 more favorably affects RuBP regen- anatomical features among three morphologically diverse crop eration than carboxylation rate. Notably, the ratio of Jmax : Vcmax types of B. rapa with past selection for different harvestable com- is significantly higher in leaf crops than oilseed types or turnips ponents (seed oil, tubers, and leaves). Differences in crop mor- (Fig. 4c; Table S3e). The high Jmax : Vcmax ratio indicates more phology that relate to size, sink strength, and/or allocation were allocation to light harvesting (increased electron transport and associated with carbon fixation and stomatal conductance and RuBP regeneration) in leaf crops, with commensurately higher with leaf histological features that affect these gas exchange traits, commitment to photosynthetic carbon metabolism (carboxyla- suggesting physiological evolution during crop diversification tion) in oilseeds. A strong negative association is observed and thus filling an important empirical knowledge gap. As a between leaf area and percentage of nitrogen (Fig. 4d), possibly novel component to crop diversification, the circadian clock also indicating higher Rubisco concentrations in the smaller oilseed exhibited correlated evolution with gas exchange traits. This cor- leaves. related evolution may reflect a response to selection during diver- Differences in CO2 supply could provide an additional expla- sification, in which crop varieties (or possibly just individual nation for higher carbon assimilation in oilseeds, that is, in addi- genotypes) with greater sink strength, such as oilseeds, are tion to more efficient photosynthetic biochemistry (as indicated selected for higher carbon fixation and a circadian period closer by Vcmax and Jmax earlier), oilseeds may have higher CO2 supply to 24 h enables higher photosynthesis. We further identified leaf- as a result of leaf morphological features. The oilseed genotypes level biochemical features that contribute to genotypic differences we sampled in fact have 40% more stomata than vegetable geno- in gas exchange. Later, we discuss the results in the light of previ- types (Fig. 5a–c; Table S3f), and stomatal density is positively ous studies examining phenotypic outputs of the clock and diver- = < correlated with stomatal conductance, gs (r 0.48, P 0.001). sification of other crop species. Further, internal leaf anatomical features, such as a thinner adaxial epidermis (Fig. 6a; Table S3 g), probably improve gas Circadian clock associations with physiological processes in supply in oilseeds. Oilseeds also tend to have thicker mesophyll crop morphotypes and more palisade layers (Fig. 6b,c; Table S3 h). Thicker palisade mesophyll is one mechanism to achieve a high Amax, because Translational research has examined clock function and the additional palisade cells provide more space for chloroplasts, light Arabidopsis thaliana clock model in crop plants. In several cases,

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Fig. 4 Response of photosynthesis to internal CO2 concentrations at 25°C and light saturation. (a) Representative CO2 response curves of photosynthesis À2 À1 at 25°C for three crop types of Brassica rapa. (b) Differences in Rubisco carboxylation rate (Vcmax)(lmol CO2 m s ) between different crop types at 25°C. (c) Differences in the ratio of the maximum electron transport rate driving ribulose-1,5-bisphosphate (RuBP) regeneration (Jmax) to the maximum RuBP carboxylation rate (Vcmax)at25°C. Error bars indicate Æ 1SE. Different letters indicate statistically significant differences among crop types (P < 0.05). (d) Association between percentage nitrogen per unit leaf mass as a function of leaf area. Each symbol represents a genotype; crop types are coded by color. The line represents the following relationship: r = À0.44 (R2 = 0.19), P = 0.0007. orthologs of A. thaliana circadian genes with conserved function c. 24 h, while the vegetable types have longer circadian cycles of are found in crop species, including Oryza (Murakami et al., 25 h, on average, for leaf crops and nearly 26 h for turnips 2003), Poplar (Takata et al., 2009), and B. rapa (Murakami et al., (Fig. 1). In combination with circadian clock differences, we 2003; Takata et al., 2009; Kim et al., 2012; Lou et al., 2012; Xie observed a significant crop type effect on both Amax and gs et al., 2015). Clock outputs documented in A. thaliana are also (Fig. 2), such that oilseeds have the highest photosynthetic rates observed in crop species. For instance, in a single genotype of a while the leaf crops have the lowest. While clock period was not legume, Phaseolus vulgaris, the circadian clock regulates photo- correlated with unadjusted values of gas exchange, we found that synthesis and stomatal conductance (Fredeen et al., 1991; Hen- circadian period and stomatal conductance were negatively corre- nessey & Field, 1991; Hennessey et al., 1993). In experimental lated after statistically factoring out differences in biochemical segregating progenies of B. rapa, quantitative clock variation (and demand, as oilseeds with shorter circadian periods near 24 h had clock quantitative trait loci) is associated with gas exchange higher rgs (Fig. 3c). After statistically accounting for genotypic (Edwards et al., 2011), and in the wild near-relative, Boechera variation in CO2 supply, circadian period and rAmax were posi- stricta, quantitative clock variation correlates with root : shoot tively correlated; this suggests that long-period vegetable types allocation and growth rates (Salmela et al., 2015). have higher rAmax than oilseeds when gas supply is factored out Here, we show that crop types of B. rapa differ significantly in (Fig. 3b). Although any mechanistic connection to the clock circadian period. The mean period for the oilseed crop type was remains to be explained, the higher adjusted values of rAmax in

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(a) (a)

(b) (b)

Fig. 6 Differences in anatomical parameters within different Brassica rapa accessions from representative cross-sections. (a) Maximum depth of adaxial epidermis; (b, c) oilseed (b) and turnip (c) cross-sections, in which differences in adaxial epidermal depth and number of palisade layers are Fig. 5 Differences in stomatal density between Brassica rapa crop À2 visible. Bars, 100 lm. Error bars indicate Æ 1 SE. Different letters indicate accessions. (a) Stomatal density (number of stomata mm ). Error bars statistically signiﬁcant differences among crop types (P < 0.05). Light indicate Æ 1 SE. Different letters indicate statistically signiﬁcant differences micrographs have had the background removed and false color applied to among B. rapa crop types (P < 0.05). (b) Stomatal density of a highlight differences in palisade parenchyma cell layers. Red lines delineate representative oilseed; (c) stomatal density of a representative cabbage. the transition from palisade parenchyma to spongy mesophyll.

leaf crops relative to oilseeds may be explained, in part, by their amounts of total biomass accumulation than oilseeds in our study higher Jmax : Vcmax; leaf crops may be more biochemically efﬁ- (Fig. 3d), suggesting that photosynthetic biochemistry of the cient in light harvesting and electron transport, while oilseeds former genotypes, allocation feedbacks on photosynthesis, and may allocate photosynthetic proteins preferentially to carbohy- possibly allocation differences (e.g. to vegetative organs rather drate metabolism to support metabolically expensive oil produc- than metabolically expensive, low-mass seed oil) may outweigh tion (see later). Differences in carbon allocation and associated their reduced CO2 supply in determining vegetative growth. feedbacks on photosynthesis (Farrar & Jones, 2000; Paul & The observed association between period and adjusted rgs is Pellny, 2003) could also contribute to higher adjusted rAmax in consistent with functional hypotheses regarding clock function, vegetable types. Long-period vegetable crops also showed higher in which period lengths close to 24 h are predicted to increase gas

New Phytologist (2015) Ó 2015 The Authors www.newphytologist.com New Phytologist Ó 2015 New Phytologist Trust New Phytologist Research 9 exchange by providing a match to the duration of daily cycles. or its activation status, or a combination of both (Quick et al., The association between period and adjusted rAmax as well as veg- 1991; Baker et al., 1997). Lower Vcmax rates are often explained etative biomass, in which cycles > 24 h correlate with increased by lower amounts of Rubisco (Jacob et al., 1995; Nakano et al., values of these two traits, requires further mechanistic investiga- 1997; Tissue et al., 1999) and sometimes by a low activation state tion; interestingly, vegetable crops of B. oleracea likewise exhibit (Sage et al., 1989; Cook et al., 1998). Usually, there is a positive longer period lengths than floral crops (A. Millar, pers. comm.). correlation between light-saturating photosynthetic rate and N Regardless of the exact mechanistic relationships, the results concentration, because N may reflect Rubisco abundance (Evans, provide a novel demonstration of correlated evolution between 1989). Consistent with this pattern, we observed that the amount the clock and gas exchange, which is potentially relevant to of N was significantly correlated with the leaf area; oilseeds have understanding the developmental origin of crop differences in smaller leaves, more nitrogen per unit leaf mass, and potentially physiological traits as well as developmental targets for crop more Rubisco. Other processes that could explain crop differ- improvement. ences in RuBP regeneration and electron transport and that war- rant further investigation include regulation of enzymatic activity of photosynthetic pigment–protein complexes as well as enzyme Gas exchange, leaf biochemistry, and yield kinetics, activation states, and activation rates that affect photo- Selection for oil production is associated with higher gas synthesis (Evans, 1987; Evans & Terashima, 1987; Bernacchi exchange rates in multiple oil crops. For instance, photosynthesis et al., 2002). affects oil production in rapeseed, B. napus, by participating in lipid metabolism and serving as a source of NADPH and ATP Leaf anatomy and gas exchange (Ruuska et al., 2004; Goffman et al., 2005; Li et al., 2006). Culti- vars of short-season soybean (Glycine max) show improvement of In several studies, leaf anatomy has been shown to affect gas oil yield coupled with an increase in photosynthetic rates and exchange performance (Reich et al., 1998; Niinemets, 1999; stomatal conductance (Morrison et al., 1999; Cober et al., 2005). Wright et al., 2004; Franks & Farquhar, 2007). The overall Oil production in Helianthus annuus is likewise positively corre- response of stomatal conductance to stomatal density is straight- lated with leaf area, which is a proxy for photosynthetic rates in forward in the absence of other morphological changes, namely some species (Herve et al., 2001). Here, higher gas exchange is an increase in stomatal density directly increases stomatal con- observed in oilseed relative to vegetable crops, and, specifically, ductance for a given set of environmental conditions (Woodward high values of gs in oilseeds support high values of Amax (Fig. 3a). & Bazzaz, 1988; Franks & Beerling, 2009). Several cultivated In all of these species, high photosynthetic rates in oilseeds might species have increased stomatal density compared with the wild be attributable to increased sink strength, because metabolically relatives (e.g. in the genera Arachis, Beta, Gossypium, and expensive oil production drives high carbohydrate demand. Phaseolus), although some domesticated species show a decrease The main biochemical processes that contribute to photosyn- in stomatal density (e.g. the genera Avena, Cichorium,andVicia) thetic performance in C3 plants are RuBP carboxylation rates (Milla et al., 2013). We found that the increase in stomatal den- (Vcmax) and how rapidly sugars are thus produced, as well as sity in oilseeds relative to vegetable crops (Fig. 5) was positively RuBP regeneration (expressed as Jmax) (Sage, 1994). The change associated with stomatal conductance. Leaf anatomical features in Jmax : Vcmax is correspondingly explained as a change in the can also affect several aspects of gas exchange. Specific mesophyll allocation of photosynthetic proteins to light harvesting or to phenotypes, for instance, can affect light absorption and carbon sugar production. Among B. rapa crop types, we observe that fixation. Higher numbers of palisade mesophyll cell layers are Vcmax values are highest among oilseeds while Jmax : Vcmax ratios associated with higher photosynthetic rates (Vogelmann & are higher among leaf crops than among either oilseeds or Evans, 2002; Terashima et al., 2006). Fig. 6 shows an oilseed – turnips. Combined with their high CO2 supply, oilseeds of genotype (159 WO-181) that has five palisade layers, while the B. rapa seem to have more favorable allocation of these proteins, vegetable turnip (189 – VT-091) has three layers, a phenotypic which leads to higher unadjusted values of Amax (Fig 2b). When pattern consistent with our observation of higher Amax in oilseeds CO2 supply is factored out, high Jmax : Vcmax values (in leaf crops) than in turnips. The uppermost 20% of the leaf absorbs light on and possible feedback from allocation (in both leaf and root the leaf surface (Terashima & Saeki, 1983; Vogelmann et al., crops) may enhance adjusted values of rAmax in vegetable relative 1989); thus, a thinner upper epidermis may be beneficial for to oilseed crops (Fig. 3b). increasing light absorption rates and CO2 fixation. We observe Patterns of carbohydrate production may be relevant to thinner adaxial epidermis in oilseeds of B. rapa, which might con- crop diversification for different targets of harvest. The product of tribute to their higher photosynthetic and stomatal conductance carboxylation of RuBP consists of two molecules of 3- rates. phosphoglycerate, which is the precursor to a range of carbohy- While past studies and the current study indicate an associa- drates that are stored and transported by other pathways. As tion between the circadian clock and gas exchange, there is no described earlier, oilseeds probably require carbohydrates as a evidence in the literature that leaf morphological and anatomi- source of energy for oil production, which corresponds with our cal traits are under circadian clock regulation. There are many result of higher Vcmax values in this crop type (Fig. 4b). Variation annotated A. thaliana genes that control leaf anatomy and in Vcmax can result from changes in Rubisco protein abundance development (e.g. SDD1, YABBY genes, PHB, PHV, and

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GTL). Using published A. thaliana transcriptome data (Coving- Cook A, Tissue D, Roberts S, Oechel W. 1998. Effects of long-term elevated [CO2] from natural CO2 springs on Nardus stricta: photosynthesis, ton et al., 2008), we examined whether A. thaliana genes that – contribute to anatomical or morphological features are under biochemistry, growth and phenology. Plant, Cell & Environment 21: 417 425. Cook M, Evans L. 1983. Some physiological aspects of the domestication and circadian control, and found that no obvious candidate genes improvement of rice. Field Crops Research 6: 219–238. involved in anatomical patterning are regulated by the circa- Covington MF, Maloof JN, Straume M, Kay SA, Harmer SL. 2008. Global dian clock. transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biology 9: R130. Dalchau N, Baek SJ, Briggs HM, Robertson FC, Dodd AN, Gardner MJ, Conclusions Stancombe MA, Haydon MJ, Stan G-B, Goncßalves JM. 2011. The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis Human selection during B. rapa diversification favored increases thaliana circadian clock to sucrose. Proceedings of the National Academy of in specific harvestable components. Our results, including PICs, Sciences, USA 108: 5104–5109. indicate that circadian period is correlated over evolutionary time Darwin C. 1868. The variation of animals and plants under domestication. with gas exchange and biomass accumulation in B. rapa. These London, UK: John Murray. De Vries I. 1997. Origin and domestication of Lactuca sativa L. Genetic Resources results are consistent with the long-standing view that targeted and Crop Evolution 44: 165–174. modification of the circadian clock could enable further crop Del Carpio DP, Basnet RK, Arends D, Lin K, De Vos RC, Muth D, Kodde J, improvement. More generally, the results suggest the hypothesis Boutilier K, Bucher J, Wang X. 2014. Regulatory network of secondary that evolution at clock loci might partly contribute to increases in metabolism in Brassica rapa: insight into the glucosinolate pathway. PLoS ONE photosynthetic rates and conductance commonly observed in 9: e107123. Diaz-Uriarte R, Garland T. 1996. Testing hypotheses of correlated evolution oilseeds of other crops (Herve et al., 2001; Ruuska et al., 2004; using phylogenetically independent contrasts: sensitivity to deviations from Cober et al., 2005). The reversal in correlation between period Brownian motion. Systematic Biology 45:27–47. and rAmax (or biomass) (Fig. 3b,d) compared with period and rgs Diaz-Uriarte R, Garland T. 1998. Effects of branch length errors on the performance of phylogenetically independent contrasts. Systematic Biology 47: (Fig. 3c) could contribute to the maintenance of clock variation – within the clade of B. rapa crop genotypes. We further observed 654 672. Dodd AN, Salathia N, Hall A, Kevei E, Toth R, Nagy F, Hibberd JM, Millar that enhanced photosynthesis is associated with leaf anatomical AJ, Webb AA. 2005. Plant circadian clocks increase photosynthesis, growth, and morphological traits in a manner consistent with other crops, survival, and competitive advantage. Science 309: 630–633. and suggest that further functional studies, including clock Edwards CE, Ewers BE, Williams DG, Xie Q, Lou P, Xu X, McClung CR, Weinig C. 2011. The genetic architecture of ecophysiological and circadian mutants in crop morphotypes, are needed to dissect the specific – genetic mechanisms at work. traits in Brassica rapa. Genetics 189: 375 390. Edwards CE, Haselhorst MS, McKnite AM, Ewers BE, Williams DG, Weinig C. 2009. Genotypes of Brassica rapa respond differently to plant-induced Acknowledgements variation in air CO2 concentration in growth chambers with standard and enhanced venting. Theoretical and Applied Genetics 119: 991–1004.

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