Shading during the grain filling period increases 2-acetyl-1-pyrroline content in fragrant rice

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1 Mo et l. Rice (2015) 8:9 DOI /s y RESEARCH Open Access Shding during the grin filling period increses 2-cetyl-1-pyrroline content in frgrnt rice Zhowen Mo 1,2,WuLi 3, Shenggng Pn 1,2, Timothy L Fitzgerld 4, Feng Xio 1, Yongjin Tng 1, Yilei Wng 1, Meiyng Dun 1,2, Hu Tin 1,2 nd Xingru Tng 1,2* Astrct Bckground: Frgrnt rice, including Thi jsmine nd Indin smti vrieties, is highly vlued y consumers glolly. 2-cetyl-1-proline (2-AP) is the mjor compound responsile for the romtic chrcter of frgrnt rice. Previously, environmentl fctors such s wter mngement nd slinity hve een proven to influence 2-AP levels in frgrnt rice; ssessing the effect of dditionl environmentl fctors on 2-AP concentrtion is therefore eminent. The level of solr rdition (solr intensity; SI) to which crop is exposed cn ffect growth, yield nd grin qulity, nd other photosynthetic nd physiologicl chrcteristics. In this study the effect of shding (i.e. the reduction of SI) on yield, qulity, nd 2-AP concentrtion in two elite Chinese frgrnt rice vrieties, Yuxingyouzhn nd Nongxing 18, hs een investigted. Furthermore, ccumultion of the plnt stress response molecules proline nd gmm-minoutyric cid, which hve lso een implicted in pthwys leding to 2-AP production, ws ssessed to study shding effects on these compounds in frgrnt rice, nd to further possily determine fluxes in iochemicl pthwys leding to 2-AP ccumultion. Results: This study hs reveled significnt chnges in the yield nd qulity chrcters under shding tretment. Additionlly, 2-AP nd GABA content in grins ws significntly incresed for ll shding tretments in oth vrieties. In ddition to 2-AP, ten other voltile compounds were studied; results indicted tht shding tretments could hve selective effect on the metolism of these voltile compounds. Conclusions: In this study, we hve demonstrted tht shding during grin filling hs significnt effects on yield nd qulity trits in rice, nd leds to the ccumultion of GABA nd 2-AP. We discuss the implictions of these findings in terms of pthwys leding to 2-AP nd GABA production in frgrnt rice, which hve not een fully elucidted. The shding effect on ten dditionl voltile compounds is lso discussed. Finlly we discuss possile effects of vrition in solr intensity resulting from nthropogenic emissions on frgrnt rice production. Keywords: 2-cetyl-1-pyrroline; Aromtic rice; γ-minoutyric cid; Proline; Shding tretment; Yield Bckground Frgrnt rice vrieties possess chrcteristic rom tht is often descried s nutty or popcorn-like (Brynt nd McClung, 2010). Thi jsmine nd indin smti re the most widely-recognized types of frgrnt rice. Frgrnt rice is highly desired y consumers nd ttrcts premium price in mny mrkets (Skthivel et l., 2009); * Correspondence: tngxr@scu.edu.cn Equl contriutors 1 College of Agriculture, South Chin Agriculturl University, Gungzhou, Gungdong, , Chin 2 Scientific Oserving nd Experimentl Sttion of Crop Cultivtion in South Chin, Ministry of Agriculture. P. R. Chin, Gungzhou, Gungdong , Chin Full list of uthor informtion is ville t the end of the rticle furthermore, glol demnd for frgrnt rice is incresing (Hshemi et l., 2013). The voltile compounds in rice re complicted; instrumentl nlyses hve oserved more thn 200 voltile compounds (Tsugit, 1985;Buttery et l., 1988; Chmpgne, 2008). Buttery et l. (1988) demonstrted tht the prole key contriutors to cooked rice rom mong the detected compounds were 2- cetyl-l-pyrroline, (E,E)-2,4-decdienl, nonnl, hexnl, (E)-2-nonenl, octnl, decnl, 4-vinyl-guicol, nd 4-vinylphenol. Jezussek et l. (2002) suggested tht 2-mino cetophenone nd 4,5-epoxy-(E)-2-decenl were importnt previously unknown rice rom compounds. Mrvl et l. (2008) indicted tht 2015 Mo et l.; licensee Springer. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly credited.

2 Mo et l. Rice (2015) 8:9 Pge 2 of 10 orgnic extrcts of cooked romtic rice showed similr rom profile etween the cultivrs, ut showed sustntil differences in the levels of vrious voltile compounds. Moreover, Yng et l. (2008) suggested tht 13 voltile coupounds my contriute to differences in rom. Despite the complexity of voltile compounds in rice (Chmpgne et l., 2005), single voltile compound, 2-cetyl-1-pyrroline (2-AP), is well-proven to e primrily responsile for the romtic chrcter of frgrnt rice (Buttery et l., 1983, 1986; Widjj et l., 1996; Jezussek et l., 2002). In frgrnt rice, 2-AP cn e detected in ll prts of the plnt except the roots (Buttery et l., 1983; Mrvl et l., 2010); 2-AP is lso present in non-frgrnt rice vrieties, ut t much lower concentrtion (Widjj et l., 1996). The inctivtion of BADH2, encoding n minoldehyde dehydrogense, is responsile for the ccumultion of 2-AP in frgrnt rice vrieties (Brdury et l., 2005; Brdury et l., 2008). Proline is precursor of 2-AP in rice (Yoshihshi et l., 2002), nd γ-minoutyrldehyde (GABld) is likely to e the direct precursor to 2-AP production vi BADH2; Brdury et l. (2008) proposed tht functionl BADH2 ctlyses the conversion of GABld to γ-minoutyric cid (GABA), while non-functionl BADH2 in frgrnt rice leds to cyclistion of GABld to Δ 1 -pyrroline, with the ddition of n cetyl group forming 2-AP. The role of proline in plnt stress tolernce is well-estlished (Szdos nd Svouré, 2010); dditionlly, GABA ccumultes in response to iotic nd iotic stresses in plnts (Bouche nd Fromm, 2004) nd is therefore speculted to ply role in stress tolernce (Kinnersley nd Turno, 2000). Thus, it ppers tht there is n overlp in pthwys involved in 2-AP ccumultion nd the response to stress. In further support of n interction etween 2-AP production nd stress responses, environmentl fctors hve een oserved to influence the level of 2-AP in frgrnt rice grins. For exmple, Yoshihshi et l. (2002) reported tht drought stress during grin formtion incresed 2-AP content, nd Gy et l. (2010) nd Poonlphdech et l. (2012) hve lso recently reported n increse in 2-AP ccumultion in response to slt stress. Interestingly, elite frgrnt rice vrieties re reltively susceptile to iotic nd iotic stresses (Niu et l., 2008), nd recently n ssocition of the frgrnce phenotype with slt susceptiility hs lso een reported (Fitzgerld et l., 2010; Wijerthn et l., 2014). Photosyntheticlly Active Rdition (PAR) is the rnge of electromgnetic rdition etween 400 nd 700 nnometers, roughly equivlent to the visile light spectrum, tht plnts (nd most photosynthetic orgnisms) hrness for photosynthesis. The level of PAR to which plnt is exposed is relted to photosynthetic rte (McCree, 1981), nd vrition in PAR cn therefore hve mjor effect on plnt growth nd development. In the field, the intensity of solr rdition (solr intensity; SI) determines the level of PAR to which crop is exposed, nd vrition in solr rdition cn ffect crop yield (Liu nd Tollenr, 2009). Seson, geogrphic loction, nd cloud cover re key fctors influencing SI; dditionlly, SI vries in response to tmospheric pollution nd wter vpour content (Jáuregui nd Luyndo, 1999; Hywood et l., 2011). The level of SI to which plnt is exposed cn lso e strongly ffected y surrounding vegettion; shding stress in plnts growing eneth or mongst cnopy, nd responses to such stress, re well-recognised phenomen (Gommers et l., 2013). To understnd how rice responds to low light environment, reserchers hve used rtificil shding to control light density. Studies hve showed tht shding cn ffect rice morphologicl chrcteristics, physiologicl chrcteristics, yield, nd qulity. For exmple, Tng (1988) found tht shding ffected growth nd yield, decresed dry mtter, nd incresed lef thickness. Ci nd Luo (1999) reported tht shding t different stges decresed rice yield, ccumultion rte of dry mtter nd the uptke of nutrients, ut incresed nutrient content. Ren et l. (2003) indicted tht the distriution nd trnsformtion of plnt dry mtter is ffected y shding, nd some shding levels decresed rice yield. Besides, Deng et l. (2009) found tht the filled grin percentge nd yield were oviously ffected y shding. Moreover, Liu et l. (2012) reported tht the effect of shding on the content of mlondildehyde, soule sugr, soule protein, nd protective enzymes ctivities in leves vrious in genotype. Ding et l. (2004) found tht shding fter heding extended rice growth durtion of the flg lef, decresed decomposing rte of chlorophyll nd MDA content, reduced the photosynthesis of the flg lef, nd tht the degree of the grinfilling ws lower thn tht of under the strong light. Zhng et l. (2007) indicted tht shding ffected rice qulity differently mongst genotypes, however shding incresed protein content nd chlkiness nd decresed mylose content of ll rice cultivrs. Liu et l. (2008) found tht wek light during grin filling ffected rice yield, physiologicl chrcteristics nd qulity, prticulrly the rte of chlky grins. Additionlly, shding hs een demonstrted to effect strch synthse nd relted enzyme ctivities (Wng et l., 2013). Previous studies hve ssessed the effects of n rtificil shding (which results in decrese in SI nd PAR) on growth nd development, yield, nd grin qulity in rice (e.g. Tnk nd Kwno, 1966; Ci nd Luo, 1999; Ren et l., 2003; Zhng et l., 2007). Here we hve studied the impct of shding during the grin filling period on GABA, proline, nd 2-AP ccumultion in frgrnt rice. Furthermore, to provide dditionl insight into the

3 Mo et l. Rice (2015) 8:9 Pge 3 of 10 effect of shding on growth nd yield in frgrnt rice, we hve complemented our nlyses of metolite ccumultion with ssessment of totl nitrogen levels, nd grin yield nd qulity in response to shding tretment. Results Effect of shding tretment on yield, yield relted trits, dry weight, nd hrvest index For Yuxingyouzhn, ll shding tretments resulted in significnt reduction in filled grin percentge, grin weight, grin yield, totl dry weight, nd hrvest index. Additionlly, shding tretment during the whole grin filling period (S1) nd during the ltter stge of grin filling (S3) cused significnt reduction of pnicle numer. For Nongxing 18, shding tretment during the whole grin filling stge S1 nd during erly grin filling (S2) cused significnt reduction of filled grin percentge nd 1000-grin weight; nd S1 significntly decresed grin yield nd totl dry weight (Tle 1). On verge, Yuxingyouzhn hd lower pnicles numer, filled grin percentge, 1000-grin weight, nd totl dry weight, ut hd higher numer of grins per pnicle, grin yield, nd hrvest index thn Nongxing 18 (Tle 1). Effect of shding tretment on grin qulity Significnt effects were identified for some shding tretments on most qulity trits. All shding tretments significntly incresed grin protein content in oth Yuxingyouzhn nd Nongxing 18 (Tle 2). Additionlly, similr effect of shding on chlkiness chrcterstics ws oserved in oth cultivrs; shding during the whole grin filling period nd during lte grin filling resulted in decrese in men rte of chlky grins, nd men degree of chlkiness, however shding during erly grin filling significntly incresed chlkiness chrcteristics (Tle 2). No significnt effect on rown rice rte ws detected in the shding tretments for either cultivr, while for milled rice rte, hed rice rte nd lkli spreding vlue effects vried with timing, during shding s well s cultivr (Tle 2). Effect of shding tretment on 2-cetyl-1-pyrroline, GABA, proline, nd totl nitrogen levels The 2-AP content in grins ws significntly incresed for ll shding tretments in oth vrieties, with increses of %, nd % detected for Yuxingyouzhn nd Nongxing 18, respectively. In oth vrieties, the highest men 2-AP content in grins ws identified for plnts exposed to S2 tretment, with 2-AP contents of μg kg 1 in Yuxingyouzhn nd μg kg 1 in Nongxing 18, compred to respective mens of nd μg kg 1 in the sence of shding (S0) (Figure 1). All shding tretments lso significntly incresed GABA in the grins of Yuxingyouzhn ( % ) nd Nongxing 18 ( %) (Figure 2). In Yuxingyouzhn, significnt increse in proline content ws identified for S1 nd S2, ut not for S3 (Figure 3). For Nongxing 18, proline content ws not significntly ffected y ny shding tretment (Figure 3). No significnt difference in totl nitrogen content in grins etween shding tretments ws oserved (Figure 4). Reltionships mong 2-Acetyl-1-pyrroline, GABA, proline, nd totl nitrogen Significnt correltions were identified etween 2-AP nd GABA in response to shding for Yuxingyouzhn ut Tle 1 Effect of shding tretment on yield, yield relted trits, totl dry weight, nd hrvest index Tretment Pnicles numer (m 2 ) Grins per pnicle Filled grin percentge (%) 1000-grin weight (g) Grin yield (g m 2 ) Totl dry weight(g m 2 ) Yuxingyouzhn Hrvest index (%) S S c c c c S S c c c men * * Nongxing 18 S S c S S men * * Mens in the sme column followed y different lower cse letters for the sme vriety differ significntly t P = 0.05 y LSD tests. Mens of the two vrieties followed y sterisk for the sme detected index difer significnt t P = 0.05 y LSD tests.

4 Mo et l. Rice (2015) 8:9 Pge 4 of 10 Tle 2 Effect of shding tretment on grin qulity Tretment Brown rice rte (%) Milled rice rte (%) Hed rice rte(%) Yuxingyouzhn Protein content(%) Amylose content(%) Alkli grins with chlkiness (%) Chlkiness degree (%) S c c S c c c 7.33 S c S d c 7.70 men 83.67* 72.77* 69.75* 9.85* 24.49* 7.45* 22.08* 10.46* Nongxing 18 S d S c 0.71 c S S c c 0.73 c men Mens in the sme column followed y different lower cse letters for the sme vriety differ significntly t P = 0.05 y LSD tests. Mens of the two vrieties followed y sterisk for the sme grin qulity trit difer significnt t P = 0.05 y LSD tests. none for Nongxing 18. No correltion ws found etween totl nitrogen nd GABA, proline or 2-AP nd etween proline nd totl nitrogen, GABA or 2-AP in either vriety (Tle 3). Effect of shding tretment on the reltive content (%) voltile compounds Eleven voltile compounds were mesured: (E)-2- Hexenl, 1-Hexnol, Heptnl, 2-cetyl-1-pyrroline, Octne, 1-Heptnol,1-Octen-3-ol, Octnl, Benzyl lcohol, Benzenecetldehyde, nd 3,8-Dimethylundecne. For Yuxingyouzhn, shding tretments significntly incresed reltive content in (E)-2-Hexenl. S3 significntly incresed reltive content in 2-AP. Shding tretments (S2 nd S3) significntly decresed reltive content in Octne. S1 tretment significntly incresed the reltive content of Octnl nd Benzyl lcohol. For Nongxing 18, shding tretments (S2 nd S3) significntly incresed the reltive content of (E)-2-Hexenl. Shding tretments significntly incresed the reltive content of 2-AP. S3 significntly decresed the reltive content of Octne, while significntly incresed the reltive content of Octnl. Shding tretment (S1 nd S3) significntly incresed the reltive content of Benzyl lcohol. Shding tretments (S1nd S2) significntly incresed the reltive content of 3,8-Dimethylundecne. As for the comprision etween the two vrieties, Yuxingyouzhn hd significnt higher reltive content of 2-AP thn Nongxing 18. The reltive content of (E)-2-Hexenl, Heptnl, Octne,nd Octnl in Nongxing 18 ws significntly higher thn tht of Yuxingyouzhn (Tle 4). Discussion Shding limits photosyntheticlly ctive rdition (PAR) nd cn e source of plnt stress. Severl studies hve ssessed the effect of shding on growth nd yield in 2-AP content µg kg c Yuxingyouzhn Nongxing 18 Figure 1 Effect of shding tretment on grin 2-AP content in grins. Verticl rs with different lower cse letters ove re significntly different t P = 0.05 y LSD tests. Cpped rs represent SD. d c S0 S1 S2 S3 GABA content µg g Yuxingyouzhn Nongxing 18 Figure 2 Effect of shding tretment on grin GABA content in grins. Verticl rs with different lower cse letters ove re significntly different t P = 0.05 y LSD tests. Cpped rs represent SD. c S0 S1 S2 S3

5 Mo et l. Rice (2015) 8:9 Pge 5 of 10 Proline content µg g c c Yuxingyouzhn Nongxing 18 Figure 3 Effect of shding tretment on proline content in grins. Verticl rs with different lower cse letters ove re significntly different t P = 0.05 y LSD tests. Cpped rs represent SD. cerel crops including rice; shding during flowering nd grin development hs een consistently found to decrese yield (e.g. Beed et l., 2007; Zhng et l., 2006; Liu nd Tollenr, 2009; Deng et l., 2009). Zhng et l. (2007) found tht shding ffects qulity trits in rice, nd generlly, shding resulted in incresed grin protein content nd decresed mylose content, s well s n incresed rte of chlky rice grins nd greter verge degree of chlkinesss. In this study, during the whole (S1), erly (S2), or lte (S3) grin filling period, shding tretment resulted in significntly decresed yield in two elite Chinese frgrnt rice vrieties, with S1 tretment resulting in the gretest yield losses. Yield losses were more sustntil in Yuxingyouzhn thn Nongxing 18 under ll shding tretments; this is consistent with previous studies in which genotype hs een found to influence yield loss due to shding in rice (Zhu et l., 2008; Deng et l., 2009). Shding tretment lso significntly ffected qulity trits. An increse in grin protein content under ll tretments nd consistent effect of tretment on grin chlkiness were oserved for oth vrieties. Significnt shding effects were lso identified for most other qulity trits ssessed, however Totl N content % Yuxingyouzhn Nongxing 18 Figure 4 Effect of shding tretment on totl nitrogen content in grins. Verticl rs with different lower cse letters ove re significntly different t P = 0.05 y LSD tests. Cpped rs represent SD. S0 S1 S2 S3 S0 S1 S2 S3 these vried with timing, durtion of tretment, nd cultivr. Together, these results re lrgely consistent with previous literture nd confirm tht shding during grin development cn hve sustntil effect on grin yield nd qulity in frgrnt rice. Here we hve found tht ll shding tretments significntly incresed the concentrtion of oth 2-AP nd GABA in the grin of Yuxingyouzhn thn Nongxing 18; furthermore, positive correltion etween the ccumultion of these compounds ws oserved in oth vrieties. Although GABA hs een shown to ccumulte in response to rnge of stresses, direct ssessment of the impct of shde on GABA ccumultion ppers limited; our results demonstrte tht the conserved response of GABA to plnt stress extends to shding in rice. Furthermore, our results indicte tht the ssocition etween plnt stress nd ccumultion of 2-AP exists for shding in ddition to drought nd slt. We lso ssessed ccumultion of proline in reponse to shding tretment. This ws significntly incresed in Yuxingyouzhn y S1 nd S2 tretment; in contrst, no significnt effect ws detected for ny shding tretment in Nongxing 18. This suggests tht there my e sustntil vrition of proline ccumultion in response to shding in frgrnt rice vrieties. Our oservtions of the ccumultion of GABA nd 2-AP in response to shding drw ttention to the hypothesized reltionship of the iochemistry of these compounds in frgrnt rice. As outlined ove, in vitro rice BADH2 ctlyses the conversion of GABld to GABA, molecule tht hs een widely implicted in plnt stress responses (Bouche nd Fromm, 2004; Kinnersley nd Turno, 2000) nd it hs een hypothesized tht in frgrnt rice non-functionl BADH2 leds to the ccumultion of GABld, which cyclises nd rects with cetyl group forming 2-AP (Brdury et l., 2008). However, lthough GABA cn e synthesized in plnts vi GABld, the primry pthwy for GABA production is thought to e the GABA shunt, in which glutmte is converted to GABA vi glutmte decroxylse (GAD) (Fit et l., 2008). If 2-AP indeed ccumultes in frgrnt rice vi pthwy tht produces GABA from GABld in the presence of functionl BADH2 (Brdury et l., 2008), it is intriguing tht the ccumultion of oth GABA nd 2-AP increses in response to shde. This suggests tht there my e prllel ctivtion of pthwys leding to GABA in response to shding in frgrnt rice, with 2-AP ccumulting from GABld rther thn GABA due to nonfunctionl BADH2, ut GABA lso ccumulting vi the GABA shunt. However, it is possile tht the ccumultion of oth GABA nd 2-AP occurs vi GABld. If nother enzyme exists in rice tht ctlyses the conversion of GABld to GABA, non-functionl BADH2 my result

6 Mo et l. Rice (2015) 8:9 Pge 6 of 10 Tle 3 Correltion coefficients mong 2-AP, GABA, totl nitrogen, nd proline content in grins Index 2-AP content Grin GABA content Proline content Totl nitrogen content Yuxingyouzhn 2-AP content ** GABA content ** Proline content Totl nitrogen content Nongxing 18 2-AP content GABA content Proline content Totl nitrogen content Significnt correltions t **p < in loss of efficiency rther thn elimintion of GABA ccumultion vi GABld, with excess GABld leding to ccumultion of 2-AP. Brdury et l., (2008) demonstrted tht BADH1, n enzyme with ~ 76% identity to BADH2, ctlyses the formtion of GABA from GABld in vitro, leit t lower efficiency thn BADH2. Furthermore, Singh et l., (2010) identified n ssocition of SNPs predicted to decrese ctivity of BADH1 on GABld with 2-AP-sed rom strength in rice. Thus, this ltter possiility seems plusile. In either cse, modultion of GABA ccumultion in response to stress seems credile explntion for the decrese in stress tolernce reported to e ssocited with frgrnce in rice (Fitzgerld et l., 2010; Wijerthn et l., 2014). For the other voltile compounds in grins, Buttery et l. (1988) demonstrted ten prole key contriutors to cooked rice rom mong the detected compounds. Two of the ten voltile compounds, 2-cetyl-l-pyrroline nd Octnl hve een studied extensively (e.g. Mhtheernont et l., 2001; Jezussek et l., 2002; Wongpornchi et l., 2003; Lohkunjit nd Noomhorm, 2004; Yng et l., 2008; Mrvl et l., 2008; Tnnuwong nd Lertsiri, 2010; Goufo et l., 2011; Goufo et l., 2010; Brynt nd McClung, 2010; Mthure et l., 2014; Mhttntwee nd Rouseff, 2014; Liynrchchi et l., 2014). In our study, 2-AP hd the highest verge reltive content mong the detected compounds in Yuxingyouzhn, while ws the the third most undnt voltile detected in Nongxing 18. Octnl hd the second higher verge reltive content mong the detected compounds in oth vrieties. There ws significnt difference in the verge verge reltive content of the two compounds etween the two vrieties. All shding tretment significntly incresed the reltive content in 2-AP in Nongxing 18, while S3 hd significnt impct on reltive content in 2-AP in Yuxingyouzhn. S1 significntly incresed the reltive content of octnl in oth vrieties. Of the compounds tht were posited to contriute to frgrnce Tle 4 Effect of shding tretment on the reltive content (%) voltile compounds in grins Compounds Yuxingyouzhn Nongxing 18 S0 S1 S2 S3 Men S0 S1 S2 S3 Men (E)-2-Hexenl 0.58c * 1-Hexnol Heptnl * 2-cetyl-1-pyrroline * Octne c 6.01c 6.68* 1-Heptnol Octen-3-ol Octnl * Benzyl lcohol Benzenecetldehyde ,8-Dimethylundecne Mens in the sme row followed y different lower cse letters for the sme vriety differ significntly t P = 0.05 y LSD tests. Mens of the two vrieties followed y sterisk for the sme compound difer significnt t P = 0.05 y LSD tests.

7 Mo et l. Rice (2015) 8:9 Pge 7 of 10 in rice y Yng et l. (2008), in ddition to 2-AP nd octnl, heptnl nd 1-octen-3-ol were lso detected in our studies; nd these two compounds hve lso een reported in other studies (Yng et l., 2008; Tnnuwong nd Lertsiri, 2010; Goufo et l., 2010; Brynt nd McClung, 2010; Mthure et l., 2014). We found tht, heptnl hd the highest verge reltive content in Nongxing 18, nd hd the second high verge reltive content in Yuxingyouzhn. Nongxing 18 hd significnt higher verge reltive content of heptnl thn Yuxingyouzhn. For other voltile compounds, (E)-2- hexenl (Buttery et l.,1988; Yng et l., 2008; Goufo et l., 2010, 2011), 1-hexnol (Mhtheernont et l., 2001; Goufo et l., 2010, 2011; Mthure et l., 2014), octne (Lohkunjit nd Noomhorm, 2004 ), 1-heptnol (Mhtheernont et l., 2001; Yng et l., 2008; Goufo et l., 2010, 2011; Liynrchchi et l., 2014), enzyl lcohol (Wongpornchi et l., 2003; Mthure et l., 2014; Liynrchchi et l., 2014), enzenecetldehyde (Lohkunjit nd Noomhorm, 2004 ), nd 3,8-dimethylundecne (Mhtheernont et l., 2001) were lso detected in our study. Shding incresed the reltive content of (E)-2-hexenl in oth vrieties. There ws no significnt difference etween shding nd non-shding tretments in 1-hexnol, 1-heptnol, nd enzenecetldehyde. Shding (S2 nd S3 for Yuxingyouzhn, S3 for Nongxing 18) decresed the reltive content of octne, nd (S1 for Yuxingyouzhn, S1 nd S3 for Nongxing 18) significntly incresed the reltive content of enzyl lcohol. The significnt difference in 3,8-dimethylundecne ws only found for Nongxing 18 in S1 nd S2 tretments. The results suggest tht shding tretments my hve selective effect on the metolism of the voltile compounds in frgrnt rice. Rice yield, grin qulity nd 2-AP content re key qulities for romtic rice. In this study, we hve ssessed the impct of shding impct on romtic rice yield, grin qulity nd 2-AP content, nd some relted trits. In ddition to providing further insight into shding effects on crop yield nd qulity, nd the interction etween 2-AP ccumultion nd stress in frgrnt rice, the results of this study my hve implictions for rice cultivtion in n environment eing drmticlly influenced y nthropogenic fctors. Air pollution cn hve shding effect, cusing sustntil decreses in surfce level solr intensity (Jáuregui nd Luyndo, 1999; Qin et l., 2006), nd Chin is well-known to experience extreme levels of ir pollution (Chn nd Yo, 2008) including in mjor griculturl regions such s the Yngtze River delt (Sho et l., 2006). There is evidence tht the impct of ir pollution hs decresed nnul solr rdition levels in Gungzhou nd even the whole South Chin region (Liu et l., 2014). Our results suggest tht high pollution levels, prticulrly during grin filling, could influence yield, qulity nd rom in elite Chinese frgrnt rice vrieties. While n incresed content of 2-AP s result of the shding effect of ir pollution my e desirle, yield loss nd loss of grin qulity would clerly e detrimentl. Conclusions Here, we hve demonstrted tht shding during grin filling hs significnt effects on yield nd qulity trits in rice, nd leds to the ccumultion of GABA nd 2-AP. These results further emphsize the importnt reltionship etween stress nd 2-AP production in frgrnt rice. Continuing dvnces in the understnding of this reltionship hve gret potentil for the optimiztion of oth rom production nd stress resistnce in elite rice vrieties. Mterils nd methods Plnt mterils nd tretment conditions Two romtic rice cultivrs, Yuxingyouzhn nd Nongxing 18 were used in this study. These re the min commercil frgrnt rice cultivrs in South Chin. Field experiment ws crried out from July to Novemer, 2013 t South Chin Agriculturl University s Experimentl Frm. This region hs humid sutropicl monsoonl climte. The field consists of sndy, lomy soil nd hs een under pddy cultivtion for mny yers. The min soil properties of the experimentl site were s follows: ph, 4.88; orgnic mtter content, 25.65%; totl N, 1.362%; totl P, 0.958%; totl K, %. The experiment ws rrnged in split-plot design with vrieties s the min plots nd shding tretment s the suplots with three duplictions. After yers of plnting, it is well-known tht the durtion of grin filling in the two vrieties employed for this study is pproximltey 30 dys. Bsed on previous studies, we found tht very strong shding level, corresponding to 90% reduction in full nturl light (s mesured y Luxmeter, model ZDS-10, Chin) resulted in sustntil increse in pest nd diseses dmge. Therefore, we employed shding level equivlent to 67% reduction of full nturl light (s mesured y, Luxmeter, model ZDS-10, Chin) creted y one lyer of lck netting. Four shding tretments were studied: (i) S0: the whole phse during grin filling without shding, tken s the control; (ii) S1: shding during the whole phse of grin filling (30 dys, from Octoer 3rd to Novemer 2nd); (iii) S2: shding during the erly phse of grin filling (15 dys, from Octoer 3rd to Octoer 18th); (iv) S3: shding during the lter phse of the grin filling (15 dys, from Octoer 18th to Novemer 2nd). The whole grin filling stge ws from the end of pollintion to grin dry-down, tht is from R5 to R9 in the report Counce et l. (2000), equivlent to out 30 dys for the two experimentl vrieties. As outlined ove, the whole grin filling stge ws devided

8 Mo et l. Rice (2015) 8:9 Pge 8 of 10 into two stges, one ws the erly phse of grin filling (15 dys), the other ws the lter phse of grin filling (15 dys). The re for ech suplot ws 16 m 2. Seeds were sown on July 15th nd trnsplnting ws performed on August 7th t density of 20 cm 20 cm (out 400 hills per plot) with two seedlings per hill. Plnts were hrvested on Novemer 5th. Fertilizer (1500 kg h 1 ; N, 12.5%; P 2 O 5, 6.0%; K 2 O, 10.0%; orgnic mtter, 15.0%) ws pplied 60% t sl, 40% t tillering stge. Irrigtion, pest nd diseses mngement, nd weed control were the sme in ll tretments following the guidelines recommended y the province. Smpling nd mesurement Determintion of dry weight Plnts from ten rndomly selected hills were tken to the lortory t mturity. Plnts were seprted into leves, (stem + sheth) nd pnicles. Smple were then oven-dried t 80 C to constnt weight for the determintion of dry weight. Determintion of totl nitrogen content Totl nitrogen content in grin ws determined s descried y Lu (1999). Briefly, the dried smples of ground grins (out 0.3 g) were digested using the H 2 SO 4 -HClO 4 method. The digestion ws then used to determine the totl nitrogen content y the Kjeldhl method with 2300 Kjeltec Anlyzer Unit (Foss Tector AB, Sweden). Determintion of yield nd yield relted trits At mturity, grin yield ws mesured from one unit smpling re (1 m 2 ) within ech plot, threshed mnully, then sun dried (djusted to moisture content of ~ 14%). Plnts from ten rndomly selected hills from ech plot were smpled to investigte yield-relted trits. Pnicle numer per m 2 ws mesured y counting the pnicle numers of ech hill within the unit re (1 m 2 )tthree different loctions in ech plot nd the men vlue ws tken s the finl result. Pnicles were threshed mnully, totl numer of grins nd numer of filled grins were counted. Five smples of 1000 grins were tken rndomly from filled seeds, weighted to record 1000-grin weight. Hrvest index ws clculted s the rtio etween grin yield nd dry weight t mturity, expressed s percentge. Determintion of grin qulity Aout 1 kg rice grin from ech tretment ws otined fter storge t room temperture for three months. Brown rice rte ws estimted using rice huller (Jingsu, Chin). Milled rice nd hed rice rtes were estimted using Jingmi testing rice grder (Zhejing, Chin). Percentge of grin with chlkiness nd chlkiness degree were estimted using n SDE-A light ox (Gungzhou, Chin). Amylose content, protein content, nd lkli of grins were determined using n Infrtec 1241 grin nlyzer (FOSS-TECATOR). Determintion of proline content, GABA content nd 2-AP concentrtion For determintion of proline, GABA, nd 2-AP, fresh smple of grins from 1 m 2 re of ech plot were hrvested nd immeditely stored t 20 C. Duplicte mesurements were tken for ech smple nd the men vlue of the three smples per tretment ws tken s the finl result. The proline content ws determined ccording to the method descried y Btes et l. (1973). Briefly, grins (out 0.3 g) ws homogenized in 5 ml of 3% sulfoslicylic cid, then cooled fter heting t oiling wter th for 10 min. Smples filtered nd two ml of the filtrte ws mixed with 3 ml of ninhydrin regent ( 1.25 g ninhydrin in 30 ml glcil cetic cid nd 20 ml 6 M phosphoric cid) nd 2 ml of glcil cetic cid. The rection mixture ws then heted t oiling wter th for 30 min nd plced in n ice th for 20 min efore eing extrcted with 4 ml of toluene. The toluene extrction ws then centrifuged t 4000 rpm for 5 min. The sornce of the red chromophore in the toluene frction ws mesured t 520 nm nd the mount of proline ws determined y comprison with stndrd curve nd expressed s μg g 1. GABA content in grin ws determined s descried y Zho et l. (2009) nd Yo et l. (2008). Briefly, grin (out 0.5 g) ws homogenized in 5 ml of 60% ethnol, treted for 4 hours in oscilltions instrument (HZS-H, Chin) using frequency of 200 oscilltions per minute. The superntnt ws then trnsferred to 5 ml centrifuge tue nd centrifuged t 8000 rpm for 3 min. 1 ml of superntnt ws dded to 10 ml tue, mixed with 0.6 ml 0.2 mol L 1 (ph 9.0) sodium tetrorte, two ml 5% toluene, nd 1 ml 7% sodium hypochlorite, then cooled fter heting t 100 C in wter th for 5 minutes. The sornce of the rection solution ws mesured t 645 nm nd the mount of GABA ws determined y comprison with stndrd curve nd expressed s μg g 1. Grin smples were evluted for 2-AP concentrtion y synchroniztion distilltion nd extrction method (SDE) comined with GCMS-QP 2010 Plus (Shimdzu Corportion, Jpn) s descried y Hung et l. (2012). Briefly, Collidine (2, 4, 6-trimethylpyridine) (Sigm, Switzerlnd), ws used s n internl stndrd. 10 g of finely ground grin ws trnsferred into 500 ml roundottom flsk contining 145 ml purified wter; 5 ml of μg ml 1 internl stndrd ws then dded. A stem

9 Mo et l. Rice (2015) 8:9 Pge 9 of 10 distilltion continuous extrction hed ws ttched to the flsk, nd the flsk ws heted t 150 C y n oil th pot, ZKYY (Gungzhou, Chin). Attched to the other hed of the stem distilltion continuous extrction instrument, diethyl ether (35 ml) ws used s the solvent in 500 ml round-ottom flsk, nd the flsk ws heted t 42 C y wter th pot, HH-2 (Jingsu, Chin). During isoltion, the stem distilltion continuous extrction ws mintined t 10 C y cold wter circultion mchine, YKKY-LX-300 (Beijing, Chin). The isoltion ws performed for 35 min. The ether extrct ws then dried over sodium sulfte, filtered (0.22 μm filter pper, Shimdzu, Jpn), nd then directly used to mesure 2-AP concentrtions with the GCMS-QP 2010 Plus method s descried in Hung et l., (2012). The GCMS- QP 2010 Plus working conditions were s followings: gs chromtogrph equipped with Restek Rxi-5 ms (Shimdzu, Jpn) silic cpillry column (30 m mm μm). The uto injector ws AOC-20i, SPL1. High purity helium gs (99.999%, Gungzhou Gses Co., LTD, Chin) ws the crrier gs t the flow rte of 2.0 ml min 1. The temperture of the GC oven ws 40 C (1 min), incresed t 2 C min 1 to 65 C nd held t 65 C for 1 min, nd then incresed to 220 C t 10 C min 1, nd held t 220 C for 10 min. The ion source temperture ws 200 C. Under these conditions, the retention time of 2-AP ws 7.5 min. 2-AP content ws expressed s μg kg 1.Thereltivecontentofthe rom compounds were identified on the sis of their mssspectrycompringthespectrwiththerecords of the NIST lirry. Sttisticl nlysis Anlysis of vrince nd correltion coefficients were performed using Sttistix 8 (Anlyticl, Tllhssee, Florid, USA). The dt were nlyzed y one-wy nlysis of vrince to ssess differences in yield, yield relted trits, totl dry weight, hrvest index, grin qulity, 2- cetyl-1-pyrroline, GABA, proline nd totl nitrogen etween tretments. Arevitions GABA: γ-minoutyric cid; 2-AP: 2-cetyl-1-pyrroline; GABld: γ-minoutyrldehyde; PAR: Photosyntheticlly Active Rdition; SI: solr intensity. Competing interests The uthors declre no potentil competing interests. Authors contriutions ZM, XT, WL nd SP designed the experiments, ZM,WL nd TLF nlyzed the dt, nd wrote the rticle, FX,YT, YW nd HT performed the trits investigtion, ZM nd MD conducted the GC-MS nlysis. All uthors red nd pproved the finl mnuscript. Acknowledgements This study ws supported y Ntionl Nturl Science Foundtion of Chin ( ), Nturl Science Foundtion of Gungdong Province ( ), Agriculturl Reserch Projects of Gungdong Province (2011AO ), nd Agriculturl Stndrdiztion Project of Gungdong Province (4100 F10003). The uthors would like to thnk Dr. Arydeep Roychoudhury nd Altfhusin B Ndf for their contriutions to the mnuscript. Author detils 1 College of Agriculture, South Chin Agriculturl University, Gungzhou, Gungdong, , Chin. 2 Scientific Oserving nd Experimentl Sttion of Crop Cultivtion in South Chin, Ministry of Agriculture. P. R. Chin, Gungzhou, Gungdong , Chin. 3 Crops Reserch Institute, Gungdong Acdemy of Agriculturl Sciences, Gungzhou, Gungdong , Chin. 4 CSIRO Agriculture Flgship, Queenslnd Bioscience Precinct, 306 Crmody Rd, St Luci, Queenslnd 4067, Austrli. Received: 15 July 2014 Accepted: 8 Jnury 2015 References Btes LS, Wldron RP, Tere ID (1973) Rpid determintion of free proline for wter stress studies. Plnt nd Soil 39: Beed FD, Pveley ND, Sylvester-Brdley R (2007) Predictility of whet growth nd yield in light-limited conditions. J Agric Sci 145(01):63 79 Bouche N, Fromm H (2004) GABA in plnts: just metolite? Trends Plnt Sci 9 (3): Brdury LMT, Fitzgerld TL, Henry RJ, Jin Q, Wters DL (2005) The gene for frgrnce in rice. 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