Abalawuli bokukhula kwezitshalo basetshenziswe njengesu lokunciphisa ukucindezeleka kokushisa ezitshalweni ezahlukene

Ukukhiqizwa kwelayisi kuyehla ngenxa yokushintsha kwesimo sezulu kanye nokuguquguquka eColombia.Abalawuli bokukhula kwezitshaloKusetshenziswe njengesu lokunciphisa ukucindezeleka kokushisa ezitshalweni ezahlukene. Ngakho-ke, inhloso yalolu cwaningo kwakuwukuhlola imiphumela yomzimba (ukuqhuba kwesisu, ukuqhuba kwesisu, okuqukethwe kwe-chlorophyll iyonke, isilinganiso se-Fv/Fm sezinhlobo ezimbili zerayisi ezithengiswayo ezibhekene nokucindezeleka kokushisa okuhlangene (amazinga okushisa aphezulu emini nasebusuku), izinga lokushisa le-canopy kanye nokuqukethwe kwamanzi okuhlobene) kanye neziguquguquko ze-biochemical (i-malondialdehyde (MDA) kanye nokuqukethwe kwe-prolinic acid). Ukuhlolwa kokuqala nokwesibili kwenziwa kusetshenziswa izitshalo zezinhlobo ezimbili zerayisi i-Federrose 67 (“F67”) kanye ne-Federrose 2000 (“F2000”), ngokulandelana. Zombili izivivinyo zahlaziywa ndawonye njengochungechunge lwezivivinyo. Ukwelashwa okusunguliwe kwakungokulandelayo: ukulawula okuphelele (AC) (izitshalo zerayisi ezikhuliswe emazingeni okushisa afanele (izinga lokushisa lasemini/ebusuku 30/25°C)), ukulawula ukucindezeleka kokushisa (SC) [izitshalo zerayisi ezibhekene nokucindezeleka kokushisa okuhlangene kuphela (40/25°C). 30°C)], kanti izitshalo zerayisi zacindezelwa futhi zafuthwa ngezilawuli zokukhula kwezitshalo (ukucindezeleka+AUX, ukucindezeleka+BR, ukucindezeleka+CK noma ukucindezeleka+GA) kabili (izinsuku ezi-5 ngaphambi nangezinsuku ezi-5 ngemva kokucindezeleka kokushisa). Ukufutha nge-SA kwandise okuqukethwe yi-chlorophyll okuphelele kwazo zombili izinhlobo (isisindo esisha sezitshalo zerayisi “F67″ kanye ne-“F2000″” kwakungu-3.25 kanye no-3.65 mg/g, ngokulandelana) uma kuqhathaniswa nezitshalo ze-SC (isisindo esisha sezitshalo “ze-F67″ sasingu-2.36 kanye no-2.56 mg). g-1)” kanye nelayisi “F2000″, ukusetshenziswa kwe-CK kwamaqabunga nakho kuthuthukise ukuhamba kwesisu kwezitshalo “ze-F2000″ zerayisi (499.25 vs. 150.60 mmol m-2 s) uma kuqhathaniswa nokulawula ukucindezeleka kokushisa. ukucindezeleka kokushisa, izinga lokushisa lomqhele wesitshalo lehla ngo-2–3 °C, futhi okuqukethwe kwe-MDA ezitshalweni kuyehla. Inkomba yokubekezelelana okuhlobene ikhombisa ukuthi ukusetshenziswa kwe-CK (97.69%) kanye ne-BR (60.73%) kwamaqabunga kungasiza ekunciphiseni inkinga yokucindezeleka kokushisa okuhlangene. ikakhulukazi ezitshalweni zerayisi ze-F2000. Ekuphetheni, ukufafaza i-BR noma i-CK kwamaqabunga kungabhekwa njengesu lezolimo lokusiza ukunciphisa imiphumela emibi yezimo zokucindezeleka kokushisa okuhlangene ekuziphatheni komzimba kwezitshalo zerayisi.
Ilayisi (i-Oryza sativa) lingelomndeni wakwaPoaceae futhi lingenye yezitshalo ezitshalwa kakhulu emhlabeni kanye nommbila nokolweni (iBajaj noMohanty, 2005). Indawo etshalwa ilayisi ingamahektha angu-617,934, kanti umkhiqizo kazwelonke ngo-2020 wawungamathani angu-2,937,840 ngesivuno esimaphakathi samathani angu-5.02/ha (iFederarroz (iFederación Nacional de Arroceros), 2021).
Ukufudumala komhlaba kuthinta izitshalo zerayisi, okuholela ezinhlotsheni ezahlukene zokucindezeleka okungenampilo njengokushisa okuphezulu kanye nezikhathi zesomiso. Ukushintsha kwesimo sezulu kubangela amazinga okushisa emhlabeni wonke ukwenyuka; Amazinga okushisa kulindeleke ukuthi akhuphuke ngo-1.0–3.7°C ekhulwini lama-21, okungakhuphula imvamisa kanye namandla okucindezeleka kokushisa. Amazinga okushisa aphezulu emvelo athinte irayisi, okubangela ukuthi isivuno sezitshalo sehle ngo-6–7%. Ngakolunye uhlangothi, ukushintsha kwesimo sezulu kuholela nasezimweni zemvelo ezingezinhle zezitshalo, njengezikhathi zesomiso esinzima noma amazinga okushisa aphezulu ezindaweni ezishisayo nezishisayo. Ngaphezu kwalokho, izehlakalo zokuguquguquka ezifana ne-El Niño zingaholela ekucindezelekeni kokushisa futhi zibhebhethekise umonakalo wezitshalo kwezinye izindawo ezishisayo. EColombia, amazinga okushisa ezindaweni ezikhiqiza irayisi kulindeleke ukuthi akhuphuke ngo-2–2.5°C ngo-2050, anciphise ukukhiqizwa kwerayisi futhi athinte ukugeleza komkhiqizo ezimakethe nasezintanjeni zokuhlinzeka.
Iningi lezitshalo zerayisi litshalwa ezindaweni lapho amazinga okushisa esondelene kakhulu nokukhula kwezitshalo (Shah et al., 2011). Kubikwe ukuthi amazinga okushisa aphakathi nendawo afanele kakhulu emini nasebusukuukukhula kwelayisi kanye nentuthukongokuvamile zingama-28°C kanye nama-22°C, ngokulandelana (Kilasi et al., 2018; Calderón-Páez et al., 2021). Amazinga okushisa angaphezu kwalawa angabangela izikhathi zokucindezeleka kokushisa okumaphakathi kuya kokukhulu phakathi nezigaba ezibucayi zokukhula kwelayisi (ukutshala, ukuhlobisa, ukuqhakaza, kanye nokugcwaliswa kwezinhlamvu), ngaleyo ndlela kuthinte kabi isivuno sokusanhlamvu. Lokhu kwehla kwesivuno kungenxa kakhulu yezikhathi ezinde zokucindezeleka kokushisa, ezithinta i-physiology yezitshalo. Ngenxa yokusebenzisana kwezici ezahlukahlukene, njengobude besikhathi sokucindezeleka kanye nokushisa okuphezulu okufinyelelwe, ukucindezeleka kokushisa kungabangela umonakalo omkhulu ongenakuguqulwa kumetabolism yezitshalo kanye nentuthuko.
Ukucindezeleka kokushisa kuthinta izinqubo ezahlukene zomzimba kanye nezamakhemikhali ezitshalweni. I-photosynthesis yamaqabunga ingenye yezinqubo ezisengozini kakhulu yokucindezeleka kokushisa ezitshalweni zerayisi, njengoba izinga le-photosynthesis lehla ngo-50% lapho amazinga okushisa ansuku zonke edlula u-35°C. Izimpendulo zomzimba zezitshalo zerayisi ziyahlukahluka kuye ngohlobo lokucindezeleka kokushisa. Isibonelo, amazinga e-photosynthesis kanye nokuhanjiswa kwesisu kuyavinjelwa lapho izitshalo zivezwa emazingeni okushisa aphezulu emini (33–40°C) noma amazinga okushisa aphezulu emini nasebusuku (35–40°C emini, 28–30°C). I-C isho ubusuku) (Lü et al., 2013; Fahad et al., 2016; Chaturvedi et al., 2017). Amazinga okushisa aphezulu ebusuku (30°C) abangela ukuvinjelwa okulinganiselwe kwe-photosynthesis kodwa andisa ukuphefumula ebusuku (Fahad et al., 2016; Alvarado-Sanabria et al., 2017). Kungakhathaliseki isikhathi sokucindezeleka, ukucindezeleka kokushisa kuthinta nokuqukethwe kwe-chlorophyll yamaqabunga, isilinganiso se-chlorophyll variable fluorescence kuya ku-maximum chlorophyll fluorescence (Fv/Fm), kanye nokusebenza kwe-Rubisco ezitshalweni zerayisi (Cao et al. 2009; Yin et al. 2010). ) Sanchez Reynoso et al., 2014).
Izinguquko ze-biochemical zingenye isici sokuzivumelanisa nezitshalo nokucindezeleka kokushisa (Wahid et al., 2007). Okuqukethwe kwe-proline kuye kwasetshenziswa njengesibonakaliso se-biochemical sokucindezeleka kwezitshalo (Ahmed noHassan 2011). I-Proline idlala indima ebalulekile ekusetshenzisweni kwezitshalo njengoba isebenza njengomthombo wekhabhoni noma i-nitrogen kanye nokuqiniswa kwe-membrane ngaphansi kwezimo zokushisa okuphezulu (Sánchez-Reinoso et al., 2014). Amazinga okushisa aphezulu nawo athinta ukuqina kwe-membrane ngokusebenzisa i-lipid peroxidation, okuholela ekwakhekeni kwe-malondialdehyde (MDA) (Wahid et al., 2007). Ngakho-ke, okuqukethwe kwe-MDA nakho kusetshenziswe ukuqonda ubuqotho besakhiwo se-membrane yeseli ngaphansi kokucindezeleka kokushisa (Cao et al., 2009; Chavez-Arias et al., 2018). Ekugcineni, ukucindezeleka kokushisa okuhlangene [37/30°C (emini/ebusuku)] kwandise iphesenti lokuvuza kwe-electrolyte kanye nokuqukethwe kwe-malondialdehyde elayisi (Liu et al., 2013).
Ukusetshenziswa kwezilawuli zokukhula kwezitshalo (ama-GR) kuye kwahlolwa ukuze kuncishiswe imiphumela emibi yokucindezeleka kokushisa, njengoba lezi zinto zihilelekile kakhulu ezimpendulweni zezitshalo noma ezindleleni zokuzivikela ngokomzimba ngokumelene nokucindezeleka okunjalo (uPeleg noBlumald, 2011; uYin et al. et al., 2011; u-Ahmed et al., 2015). Ukusetshenziswa kwezinsiza zofuzo ngaphandle kwezwe kube nomthelela omuhle ekubekezeleleni ukucindezeleka kokushisa ezitshalweni ezahlukene. Izifundo zibonise ukuthi ama-phytohormones afana ne-gibberellins (GA), ama-cytokinins (CK), ama-auxins (AUX) noma ama-brassinosteroids (BR) aholela ekwandeni kwezinguquko ezahlukene zomzimba kanye ne-biochemical (uPeleg noBlumald, 2011; uYin et al. Ren, 2011; uMitler et al., 2012; uZhou et al., 2014). EColombia, ukusetshenziswa kwezinsiza zofuzo ngaphandle kwezwe kanye nomthelela wazo ezitshalweni zelayisi akukaqondwa ngokugcwele futhi kwafundwa. Kodwa-ke, ucwaningo lwangaphambilini lukhombisile ukuthi ukufafaza i-BR ngamahlamvu kungathuthukisa ukubekezelela irayisi ngokuthuthukisa izici zokushintshana kwegesi, i-chlorophyll noma okuqukethwe kwe-proline kwamaqabunga ezithombo zerayisi (Quintero-Calderón et al., 2021).
Ama-Cytokinins aqondisa izimpendulo zezitshalo ekucindezelekeni okungenampilo, okuhlanganisa nokucindezeleka kokushisa (Ha et al., 2012). Ngaphezu kwalokho, kubikwe ukuthi ukusetshenziswa kwe-CK ngaphandle kunganciphisa umonakalo wokushisa. Isibonelo, ukusetshenziswa kwe-zeatin ngaphandle kwandise izinga le-photosynthetic, okuqukethwe kwe-chlorophyll a kanye ne-b, kanye nokusebenza kahle kokuthutha kwama-electron ku-creeping bentgrass (Agrotis estolonifera) ngesikhathi sokucindezeleka kokushisa (Xu noHuang, 2009; Jespersen noHuang, 2015). Ukusetshenziswa kwe-zeatin ngaphandle kungathuthukisa nomsebenzi we-antioxidant, kuthuthukise ukwakheka kwamaprotheni ahlukahlukene, kunciphise umonakalo we-reactive oxygen species (ROS) kanye nokukhiqizwa kwe-malondialdehyde (MDA) ezicutshini zezitshalo (Chernyadyev, 2009; Yang et al., 2009). , 2016; Kumar et al., 2020).
Ukusetshenziswa kwe-gibberellic acid nakho kubonise impendulo enhle ekucindezelekeni kokushisa. Izifundo zikhombisile ukuthi i-GA biosynthesis iqondisa izindlela ezahlukene ze-metabolic futhi ikhulisa ukubekezelelana ngaphansi kwezimo zokushisa eziphakeme (Alonso-Ramirez et al. 2009; Khan et al. 2020). U-Abdel-Nabi et al. (2020) bathole ukuthi ukufafaza i-GA yangaphandle (25 noma 50 mg*L) kungandisa izinga le-photosynthesis kanye nomsebenzi we-antioxidant ezitshalweni ze-orange ezicindezelwe ukushisa uma kuqhathaniswa nezitshalo ezilawulayo. Kuye kwaphawulwa nokuthi ukusetshenziswa kwe-HA yangaphandle kwandisa okuqukethwe komswakama okuhlobene, okuqukethwe kwe-chlorophyll kanye ne-carotenoid futhi kunciphisa i-lipid peroxidation ku-date palm (Phoenix dactylifera) ngaphansi kokucindezeleka kokushisa (Khan et al., 2020). I-Auxin nayo idlala indima ebalulekile ekulawuleni izimpendulo zokukhula eziguquguqukayo ezimweni zokushisa okuphezulu (Sun et al., 2012; Wang et al., 2016). Lo mlawuli wokukhula usebenza njengophawu lwe-biochemical ezinqubweni ezahlukahlukene njengokwenziwa kwe-proline noma ukuwohloka ngaphansi kokucindezeleka okungenabungozi (Ali et al. 2007). Ngaphezu kwalokho, i-AUX iphinde ithuthukise umsebenzi we-antioxidant, okuholela ekunciphiseni kwe-MDA ezitshalweni ngenxa yokuncipha kwe-lipid peroxidation (Bielach et al., 2017). USergeev et al. (2018) baqaphele ukuthi ezitshalweni ze-pea (Pisum sativum) ngaphansi kokucindezeleka kokushisa, okuqukethwe kwe-proline - dimethylaminoethoxycarbonylmethyl)naphthylchloromethyl ether (TA-14) kuyanda. Kulolu cwaningo olufanayo, baphinde babona amazinga aphansi e-MDA ezitshalweni eziphathwe kahle uma kuqhathaniswa nezitshalo ezingelashwanga nge-AUX.
Ama-Brassinosteroids ayisigaba sokulawula ukukhula esisetshenziselwa ukunciphisa imiphumela yokucindezeleka kokushisa. U-Ogweno et al. (2008) babike ukuthi i-BR spray yangaphandle yandisa izinga le-photosynthetic, i-stomatal conductance kanye nezinga eliphezulu le-Rubisco carboxylation yezitshalo zikatamatisi (Solanum lycopersicum) ngaphansi kokucindezeleka kokushisa izinsuku ezingu-8. Ukufutha ama-epibrassinosteroids ngamahlamvu kungandisa izinga le-photosynthetic lezitshalo zikakhukhamba (Cucumis sativus) ngaphansi kokucindezeleka kokushisa (Yu et al., 2004). Ngaphezu kwalokho, ukusetshenziswa kwe-BR ngaphandle kwamangqamuzana kubambezela ukuwohloka kwe-chlorophyll futhi kwandisa ukusebenza kahle kokusetshenziswa kwamanzi kanye nesivuno esiphezulu se-PSII photochemistry ezitshalweni ezingaphansi kokucindezeleka kokushisa (Holá et al., 2010; Toussagunpanit et al., 2015).
Ngenxa yokushintsha kwesimo sezulu nokuhlukahluka, izitshalo zelayisi zibhekana nezikhathi zamazinga okushisa aphezulu nsuku zonke (Lesk et al., 2016; Garcés, 2020; Federarroz (Federación Nacional de Arroceros), 2021). Ku-phenotyping yezitshalo, ukusetshenziswa kwe-phytonutrients noma ama-biostimulants kuye kwafundwa njengesu lokunciphisa ukucindezeleka kokushisa ezindaweni ezitshala irayisi (Alvarado-Sanabria et al., 2017; Calderón-Páez et al., 2021; Quintero-Calderón et al., 2021). Ngaphezu kwalokho, ukusetshenziswa kwezinto eziphilayo kanye neziguquguqukayo (izinga lokushisa lamaqabunga, ukuhanjiswa kwesisu, amapharamitha e-chlorophyll fluorescence, i-chlorophyll kanye nokuqukethwe kwamanzi okuhlobene, i-malondialdehyde kanye nokuhlanganiswa kwe-proline) kuyithuluzi elithembekile lokuhlola izitshalo zerayisi ngaphansi kokucindezeleka kokushisa endaweni nakwamanye amazwe (Sánchez -Reynoso et al., 2014; Alvarado-Sanabria et al., 2017; Kodwa-ke, ucwaningo ngokusetshenziswa kwezifutho ze-phytohormonal ze-foliar elayisi ezingeni lendawo luhlala lungavamile. Ngakho-ke, ucwaningo lokusabela komzimba kanye ne-biochemical kokusetshenziswa kwabalawuli bokukhula kwezitshalo lubaluleke kakhulu esiphakamisweni samasu asebenzayo ezolimo kulokhu. ukubhekana nemiphumela emibi yesikhathi sokucindezeleka kokushisa okuyinkimbinkimbi elayisini. Ngakho-ke, inhloso yalolu cwaningo kwakuwukuhlola imiphumela yomzimba (ukuhanjiswa kwesisu, amapharamitha e-chlorophyll fluorescence kanye nokuqukethwe kwamanzi okuhlobene) kanye nemiphumela ye-biochemical yokusetshenziswa kwamaqabunga kwabalawuli bokukhula kwezitshalo abane (AUX, CK, GA kanye ne-BR). (Ama-pigment e-photosynthetic, i-malondialdehyde kanye nokuqukethwe kwe-proline) Izinhlobo ezimbili zerayisi ezithengiswayo ezibhekene nokucindezeleka kokushisa okuhlangene (amazinga okushisa aphezulu emini/ebusuku).
Kulolu cwaningo, kwenziwa izivivinyo ezimbili ezizimele. Izinhlobo ze-genotypes i-Federrose 67 (F67: uhlobo lwe-genotype oluthuthukiswe emazingeni okushisa aphezulu phakathi neminyaka eyishumi edlule) kanye ne-Federrose 2000 (F2000: uhlobo lwe-genotype oluthuthukiswe eminyakeni eyishumi yokugcina yekhulu lama-20 olubonisa ukumelana negciwane lamaqabunga amhlophe) zasetshenziswa okokuqala. imbewu. kanye nokuhlolwa kwesibili, ngokulandelana. Zombili izinhlobo ze-genotypes zitshalwa kabanzi ngabalimi baseColombia. Imbewu yahlwanyelwa emathileyini angu-10-L (ubude obungu-39.6 cm, ububanzi obungu-28.8 cm, ukuphakama okungu-16.8 cm) aqukethe inhlabathi yesihlabathi esino-2% wezinto eziphilayo. Imbewu ezinhlanu ezitshalwe kusengaphambili zatshalwa ethileyi ngayinye. Ama-pallet abekwa endaweni eluhlaza ye-Faculty of Agricultural Sciences ye-National University of Colombia, i-Bogotá campus (43°50′56″ N, 74°04′051″ W), endaweni ephakeme ngamamitha angu-2556 ngaphezu kolwandle (asl). m.) futhi kwenziwa kusukela ngo-Okthoba kuya kuDisemba 2019. Ukuhlolwa okukodwa (iFederroz 67) kanye nokuhlolwa kwesibili (iFederroz 2000) ngesizini efanayo ka-2020.
Izimo zemvelo endaweni eluhlaza okotshani ngesikhathi ngasinye sokutshala zimi kanje: izinga lokushisa lasemini nasebusuku 30/25°C, umswakama ohlobene 60~80%, isikhathi se-photoperiod yemvelo amahora ayi-12 (imisebe esebenzayo ye-photosynthetically 1500 µmol (photons) m-2 s-). 1 emini). Izitshalo zavundiswa ngokokuqukethwe kwento ngayinye ezinsukwini ezingama-20 ngemva kokuvela kwembewu (DAE), ngokusho kukaSánchez-Reinoso et al. (2019): 670 mg nitrogen ngesitshalo, 110 mg phosphorus ngesitshalo, 350 mg potassium ngesitshalo, 68 mg calcium ngesitshalo, 20 mg magnesium ngesitshalo, 20 mg sulfur ngesitshalo, 17 mg silicon ngesitshalo. Izitshalo ziqukethe 10 mg boron ngesitshalo, 17 mg copper ngesitshalo, kanye 44 mg zinc ngesitshalo. Izitshalo zelayisi zagcinwa zifinyelela ku-47 DAE ekuhlolweni ngakunye lapho zifika esigabeni se-phenological V5 phakathi nalesi sikhathi. Izifundo zangaphambilini zikhombisile ukuthi lesi sigaba se-phenological yisikhathi esifanele sokwenza izifundo zokucindezeleka kokushisa elayisini (Sánchez-Reinoso et al., 2014; Alvarado-Sanabria et al., 2017).
Kuwo wonke umhloliso, kwenziwa izinhlelo ezimbili ezihlukene zokulawula ukukhula kwamaqabunga. Isethi yokuqala yezifutho ze-phytohormone zamaqabunga yasetshenziswa ezinsukwini ezi-5 ngaphambi kokwelashwa kokucindezeleka kokushisa (42 DAE) ukulungiselela izitshalo ukucindezeleka kwemvelo. Isifutho sesibili samaqabunga sabe sesinikezwa izinsuku ezi-5 ngemuva kokuba izitshalo zivezwe ezimweni zokucindezeleka (52 DAE). Kwasetshenziswa ama-phytohormone amane futhi izakhiwo zesithako ngasinye esisebenzayo esifuthwe kulolu cwaningo zibhalwe kuThebula Elingeziwe 1. Ukugxila kwezilawuli zokukhula kwamaqabunga ezisetshenzisiwe kwakungokulandelayo: (i) I-Auxin (1-naphthylacetic acid: NAA) ekugxilweni okungu-5 × 10−5 M (ii) 5 × 10–5 M gibberellin (gibberellic acid: NAA); GA3); (iii) I-Cytokinin (i-trans-zeatin) 1 × 10-5 M (iv) Ama-Brassinosteroids [i-Spirostan-6-one, 3,5-dihydroxy-, (3b,5a,25R)] 5 × 10-5; M. Lokhu kugxila kukhethwe ngoba kubangela izimpendulo ezinhle futhi kwandisa ukumelana kwezitshalo ekucindezelekeni kokushisa (Zahir et al., 2001; Wen et al., 2010; El-Bassiony et al., 2012; Salehifar et al., 2017). Izitshalo zerayisi ezingenazo izifutho zokulawula ukukhula kwezitshalo zaphathwa ngamanzi acwengekile kuphela. Zonke izitshalo zerayisi zafuthwa ngesifutho sesandla. Faka i-20 ml H2O esitshalweni ukuze umanzise izindawo ezingaphezulu nezingezansi zamaqabunga. Zonke izifutho ze-foliar zasebenzisa i-adjuvant yezolimo (i-Agrotin, i-Bayer CropScience, i-Colombia) ku-0.1% (v/v). Ibanga phakathi kwebhodwe nesifutho lingu-30 cm.
Ukwelashwa ngokucindezeleka kokushisa kwenziwa ezinsukwini ezi-5 ngemuva kokufuthwa kokuqala kwamaqabunga (47 DAE) ekuhlolweni ngakunye. Izitshalo zerayisi zadluliselwa kusuka ku-greenhouse zayiswa ekamelweni lokukhula elingu-294 L (MLR-351H, Sanyo, IL, USA) ukuze kutholakale ukucindezeleka kokushisa noma kugcinwe izimo ezifanayo zemvelo (47 DAE). Ukwelashwa ngokucindezeleka kokushisa okuhlanganisiwe kwenziwa ngokubeka igumbi emazingeni okushisa alandelayo emini/ebusuku: izinga lokushisa eliphezulu lasemini [40°C amahora ama-5 (kusukela ngo-11:00 kuya ku-16:00)] kanye nesikhathi sasebusuku [30°C amahora ama-5]. Izinsuku eziyi-8 zilandelana (kusukela ngo-19:00 kuya ku-24:00). Izinga lokushisa lokucindezeleka kanye nesikhathi sokuvezwa kwakhethwa ngokusekelwe ezifundweni zangaphambilini (Sánchez-Reynoso et al. 2014; Alvarado-Sanabía et al. 2017). Ngakolunye uhlangothi, iqembu lezitshalo ezidluliselwe ekamelweni lokukhula lagcinwa endlini eluhlaza okwesibhakabhaka ekushiseni okufanayo (30°C emini/25°C ebusuku) izinsuku ezingu-8 zilandelana.
Ekupheleni kokuhlolwa, kutholakale amaqembu okwelapha alandelayo: (i) isimo sokushisa kokukhula + ukusetshenziswa kwamanzi acwecwe [Ukulawula okuphelele (AC)], (ii) isimo sokucindezeleka kokushisa + ukusetshenziswa kwamanzi acwecwe [Ukulawula ukucindezeleka kokushisa (SC)], (iii) izimo isimo sokucindezeleka kokushisa + isicelo se-auxin (AUX), (iv) isimo sokucindezeleka kokushisa + isicelo se-gibberellin (GA), (v) isimo sokucindezeleka kokushisa + isicelo se-cytokinin (CK), kanye (vi) isimo sokucindezeleka kokushisa + i-brassinosteroid (BR) Isithasiselo. La maqembu okwelapha asetshenziselwa izinhlobo ezimbili ze-genotype (F67 kanye ne-F2000). Zonke izindlela zokwelapha zenziwa ngendlela engahleliwe ngokuphelele eneziphindaphindo ezinhlanu, ngayinye equkethe isitshalo esisodwa. Isitshalo ngasinye sasetshenziswa ukufunda iziguquguquko ezinqunywe ekupheleni kokuhlolwa. Ukuhlolwa kwathatha ama-DAE angu-55.
Ukuhanjiswa kwesisu (gs) kukalwe kusetshenziswa i-porosometer ephathekayo (SC-1, METER Group Inc., USA) esukela ku-0 kuya ku-1000 mmol m-2 s-1, enesikhala sekamelo lesampula esingu-6.35 mm. Ukulinganisa kuthathwa ngokunamathisela i-stomameter probe eqabungeni elivuthiwe kanye nehlumela eliyinhloko lesitshalo elinwetshiwe ngokugcwele. Kuyo yonke indlela yokwelapha, ukufundwa kwe-gs kwathathwa emaqabunga amathathu esitshalo ngasinye phakathi kuka-11:00 no-16:00 futhi kwalinganiswa ngokwesilinganiso.
I-RWC yanqunywa ngokwendlela echazwe nguGhoulam et al. (2002). Ishidi elandiswe ngokuphelele elisetshenziswe ukunquma i-g lasetshenziswa futhi ukukala i-RWC. Isisindo esisha (FW) sanqunywa ngokushesha ngemva kokuvuna kusetshenziswa isikali sedijithali. Amaqabunga abe esefakwa esitsheni sepulasitiki esigcwele amanzi bese eshiywa ebumnyameni ekushiseni kwegumbi (22°C) amahora angu-48. Bese ukala esikalini sedijithali bese uqopha isisindo esandisiwe (TW). Amaqabunga avuvukele omiswa kuhhavini ku-75°C amahora angu-48 bese isisindo sawo esomile (DW) saqoshwa.
Okuqukethwe kwe-chlorophyll okuhlobene kunqunywe kusetshenziswa imitha ye-chlorophyll (atLeafmeter, FT Green LLC, USA) futhi kwavezwa kumayunithi e-atLeaf (Dey et al., 2016). Ukufundwa kokusebenza kahle kwe-PSII maximum quantum (Fv/Fm ratio) kwaqoshwa kusetshenziswa i-continuous excitation chlorophyll fluorimeter (Handy PEA, Hansatech Instruments, UK). Amaqabunga aguqulwa abe mnyama kusetshenziswa ama-leaf clamps imizuzu engama-20 ngaphambi kokulinganiswa kwe-Fv/Fm (Restrepo-Diaz noGarces-Varon, 2013). Ngemva kokuba amaqabunga esejwayele ukuba mnyama, kwalinganiswa i-baseline (F0) kanye ne-maximum fluorescence (Fm). Kusukela kule datha, i-fluorescence eguquguqukayo (Fv = Fm – F0), isilinganiso se-fluorescence eguquguqukayo kuya ku-fluorescence ephezulu (Fv/Fm), isivuno esiphezulu se-quantum se-PSII photochemistry (Fv/F0) kanye nesilinganiso se-Fm/F0 kubalwe (Baker, 2008; Lee et al. ., 2017). Ukufundwa kwe-fluorescence ye-chlorophyll kanye ne-chlorophyll okuhlobene kuthathwe emaqabungeni afanayo asetshenziselwa ukulinganisa i-gs.
Cishe u-800 mg wesisindo esisha samaqabunga aqoqwe njengezinto eziguquguqukayo ze-biochemical. Amasampula amaqabunga abe esehlanganiswa ku-nitrogen ewuketshezi futhi agcinwa ukuze kuhlaziywe kabanzi. Indlela ye-spectrometric esetshenziswa ukulinganisa okuqukethwe kwe-chlorophyll a, b kanye ne-carotenoid yezicubu isekelwe endleleni kanye nezibalo ezichazwe nguWellburn (1994). Amasampula ezicubu zamaqabunga (30 mg) aqoqwa futhi ahlanganiswa ku-3 ml we-acetone engu-80%. Amasampula abe esefakwa ku-centrifuge (model 420101, Becton Dickinson Primary Care Diagnostics, USA) ngo-5000 rpm imizuzu eyi-10 ukuze kususwe izinhlayiya. I-supernatant yancishiswa yaba yivolumu yokugcina engu-6 ml ngokungeza i-acetone engu-80% (Sims and Gamon, 2002). Okuqukethwe kwe-chlorophyll kutholakale ku-663 (chlorophyll a) kanye no-646 (chlorophyll b) nm, kanye nama-carotenoid ku-470 nm kusetshenziswa i-spectrophotometer (Spectronic BioMate 3 UV-vis, Thermo, USA).
Indlela ye-thiobarbituric acid (TBA) echazwe nguHodges et al. (1999) yasetshenziswa ukuhlola i-membrane lipid peroxidation (MDA). Cishe u-0.3 g wezicubu zamaqabunga nawo wahlanganiswa ku-nitrogen ewuketshezi. Amasampula afakwa ku-centrifuge ku-5000 rpm futhi ukumuncwa kwalinganiswa ku-spectrophotometer ku-440, 532 kanye no-600 nm. Ekugcineni, ukuhlushwa kwe-MDA kwabalwa kusetshenziswa i-extinction coefficient (157 M mL−1).
Okuqukethwe kwe-proline kwazo zonke izindlela zokwelapha kunqunywe kusetshenziswa indlela echazwe nguBates et al. (1973). Engeza u-10 ml wesisombululo samanzi esingu-3% se-sulfosalicylic acid kusampula egciniwe bese ukuhlunga ngephepha lokuhlunga le-Whatman (No. 2). Ngemuva kwalokho u-2 ml walesi sihlungi waphendulwa nge-2 ml ye-ninhydric acid kanye no-2 ml we-glacial acetic acid. Ingxube yafakwa emanzini okugeza ku-90°C ihora eli-1. Misa ukusabela ngokufaka eqhweni. Shukumisa ithubhu ngamandla usebenzisa i-vortex shaker bese uncibilikisa isisombululo esiphumela ku-4 ml ye-toluene. Ukufundwa kokumuncwa kwanqunywa ku-520 nm kusetshenziswa i-spectrophotometer efanayo esetshenziselwa ukulinganisa ama-pigment e-photosynthetic (Spectronic BioMate 3 UV-Vis, Thermo, Madison, WI, USA).
Indlela echazwe nguGerhards et al. (2016) yokubala izinga lokushisa le-canopy kanye ne-CSI. Izithombe ezishisayo zithathwe ngekhamera ye-FLIR 2 (FLIR Systems Inc., eBoston, MA, e-USA) ngokunemba okungu-±2°C ekupheleni kwesikhathi sokucindezeleka. Beka indawo emhlophe ngemuva kwesitshalo ukuze kuthathwe izithombe. Futhi, amafektri amabili abhekwa njengamamodeli okubhekisela kuwo. Izitshalo zabekwa endaweni emhlophe; enye yagcotshwa nge-adjuvant yezolimo (Agrotin, Bayer CropScience, Bogotá, Colombia) ukuze ilingise ukuvulwa kwazo zonke izitshalo [imodi emanzi (Twet)], kanti enye yayiyiqabunga elingenayo ukusetshenziswa [Imodi eyomile (Tdry)] (Castro -Duque et al., 2020). Ibanga phakathi kwekhamera nebhodwe ngesikhathi sokuqopha kwakungu-1 m.
Inkomba yokubekezelelana okuhlobene ibalwa ngokungaqondile kusetshenziswa ukuqhutshwa kwe-stomatal (gs) yezitshalo eziphathwe kuqhathaniswa nezitshalo zokulawula (izitshalo ezingenazo izindlela zokwelapha ukucindezeleka kanye nezilawuli zokukhula ezisetshenzisiwe) ukuze kunqunywe ukubekezelelana kwezinhlobo ze-genotype eziphathwe ezihlolwe kulolu cwaningo. I-RTI itholakale kusetshenziswa i-equation eguqulwe ku-Chávez-Arias et al. (2020).
Kuwo wonke umvivinyo, zonke iziguquguquko zomzimba okukhulunywe ngazo ngenhla zanqunywa futhi zaqoshwa ku-55 DAE kusetshenziswa amaqabunga anwetshwe ngokuphelele aqoqwe kusuka phezulu. Ngaphezu kwalokho, izilinganiso zenziwa ekamelweni lokukhula ukuze kugwenywe ukushintsha izimo zemvelo lapho izitshalo zikhula khona.
Idatha evela ekuhlolweni kokuqala nokwesibili yahlaziywa ndawonye njengochungechunge lwezivivinyo. Iqembu ngalinye lokuhlola lalakhiwe izitshalo ezi-5, futhi isitshalo ngasinye sakha iyunithi yokuhlola. Ukuhlaziywa kokuhlukahluka (i-ANOVA) kwenziwa (P ≤ 0.05). Lapho kutholakala umehluko obalulekile, ukuhlolwa kokuqhathanisa kwe-post hoc kukaTukey kwasetshenziswa ku-P ≤ 0.05. Sebenzisa umsebenzi we-arcsine ukuguqula amanani amaphesenti. Idatha yahlaziywa kusetshenziswa isofthiwe ye-Statistix v 9.0 (i-Analytical Software, i-Tallahassee, i-FL, i-USA) futhi yadwetshwa kusetshenziswa i-SigmaPlot (inguqulo 10.0; i-Systat Software, i-San Jose, i-CA, i-USA). Ukuhlaziywa kwezingxenye eziyinhloko kwenziwa kusetshenziswa isofthiwe ye-InfoStat 2016 (i-Analysis Software, i-National University of Cordoba, i-Argentina) ukuthola abalawuli bokukhula kwezitshalo abangcono kakhulu abasacwaningwa.
Ithebula 1 lifingqa i-ANOVA ekhombisa izivivinyo, izindlela zokwelapha ezahlukene, kanye nokusebenzisana kwazo nama-pigment e-photosynthetic amaqabunga (i-chlorophyll a, b, i-total, kanye nama-carotenoid), i-malondialdehyde (MDA) kanye nokuqukethwe kwe-proline, kanye nokuhanjiswa kwe-stomatal. Umphumela we-gs, okuqukethwe kwamanzi okuhlobene. (RWC), okuqukethwe kwe-chlorophyll, amapharamitha e-chlorophyll alpha fluorescence, izinga lokushisa lomqhele (PCT) (°C), inkomba yokucindezeleka kwezitshalo (CSI) kanye nenkomba yokubekezelelana okuhlobene kwezitshalo zerayisi ku-55 DAE.
Ithebula 1. Isifinyezo sedatha ye-ANOVA mayelana nokuguquguquka kwelayisi kwezinto eziphilayo kanye nezamakhemikhali phakathi kokuhlolwa (izinhlobo zezakhi zofuzo) kanye nokwelashwa kokucindezeleka kokushisa.
Umehluko (P≤0.01) ekusebenzelaneni kwe-photosynthesis pigment yamaqabunga, okuqukethwe kwe-chlorophyll okuhlobene (ukufundwa kwe-Atleaf), kanye nemingcele ye-alpha-chlorophyll fluorescence phakathi kokuhlolwa kanye nokwelashwa kukhonjisiwe kuThebula 2. Amazinga okushisa aphezulu emini nasebusuku akhuphule okuqukethwe kwe-chlorophyll kanye ne-carotenoid. Izithombo zelayisi ezingenayo i-spray yama-phytohormones amaqabunga (2.36 mg g-1 ye-"F67″ kanye ne-2.56 mg g-1 ye-"F2000″) uma kuqhathaniswa nezitshalo ezikhuliswe ngaphansi kwezimo zokushisa ezifanele (2.67 mg g -1)) zibonise okuqukethwe kwe-chlorophyll okuphansi. Kuzo zombili izivivinyo, i-"F67" yayingu-2.80 mg g-1 kanye ne-"F2000" yayingu-2.80 mg g-1. Ngaphezu kwalokho, izithombo zerayisi eziphathwe ngenhlanganisela ye-AUX kanye ne-GA sprays ngaphansi kokucindezeleka kokushisa nazo zibonise ukwehla kokuqukethwe kwe-chlorophyll kuzo zombili izinhlobo ze-genotype (AUX = 1.96 mg g-1 kanye ne-GA = 1.45 mg g-1 ye-“F67”; AUX = 1.96 mg g-1 kanye ne-GA = 1.45 mg g-1 ye-“F67″; AUX = 2.24 mg) g-1 kanye ne-GA = 1.43 mg g-1 (ye-“F2000″) ngaphansi kwezimo zokucindezeleka kokushisa. Ngaphansi kwezimo zokucindezeleka kokushisa, ukwelashwa kwamaqabunga nge-BR kuholele ekwandeni okuncane kwalokhu kuguquguquka kuzo zombili izinhlobo ze-genotype. Ekugcineni, i-CK foliar spray ibonise amanani aphezulu kakhulu e-photosynthetic pigment ​​phakathi kwazo zonke izindlela zokwelapha (AUX, GA, BR, SC kanye ne-AC treatments) kuzinhlobo ze-genotypes F67 (3.24 mg g-1) kanye ne-F2000 (3.65 mg g-1). Okuqukethwe okuhlobene kwe-chlorophyll (iyunithi ye-Atleaf) nakho kwehlisiwe ngokucindezeleka kokushisa okuhlangene. Amanani aphezulu kakhulu aqoshwe ezitshalweni ezifuthwe nge-CC kuzo zombili izinhlobo ze-genotype (41.66 ye-“F67” kanye ne-49.30 ye-“F2000”). Izilinganiso ze-Fv kanye ne-Fv/Fm zibonise umehluko omkhulu phakathi kokwelashwa kanye nezinhlobo zezitshalo (Ithebula 2). Sekukonke, phakathi kwalezi ziguquguquko, i-cultivar F67 yayingathinteki kakhulu ekucindezelweni kokushisa kune-cultivar F2000. Izilinganiso ze-Fv kanye ne-Fv/Fm zahlupheka kakhulu ekuhlolweni kwesibili. Izithombo ze-'F2000′ ezicindezelwe ezingafafazwanga nganoma yimaphi ama-phytohormones zazinamanani aphansi kakhulu e-Fv (2120.15) kanye nezilinganiso ze-Fv/Fm (0.59), kodwa ukufafaza nge-CK kwasiza ekubuyiseleni la manani (Fv: 2591, 89, isilinganiso se-Fv/Fm: 0.73). , kuthola ukufundwa okufana nalokho okuqoshwe ezitshalweni ze-“F2000” ezikhuliswe ngaphansi kwezimo zokushisa ezifanele (Fv: 2955.35, isilinganiso se-Fv/Fm: 0.73:0.72). Kwakungekho mehluko obalulekile ku-initial fluorescence (F0), i-maximum fluorescence (Fm), i-maximum photochemical quantum yield ye-PSII (Fv/F0) kanye ne-Fm/F0 ratio. Ekugcineni, i-BR ibonise ukuthambekela okufanayo njengoba kubonwe nge-CK (Fv 2545.06, isilinganiso se-Fv/Fm 0.73).
Ithebula 2. Umphumela wokucindezeleka kokushisa okuhlangene (40°/30°C emini/ebusuku) kuma-pigment e-photosynthetic amaqabunga [i-chlorophyll iyonke (i-Chl Total), i-chlorophyll a (i-Chl a), i-chlorophyll b (i-Chl b) kanye nama-carotenoids umphumela we-Cx+c ], okuqukethwe kwe-chlorophyll okuhlobene (iyunithi ye-Atliff), amapharamitha e-chlorophyll fluorescence (i-fluorescence yokuqala (F0), i-fluorescence ephezulu (Fm), i-fluorescence eguquguqukayo (Fv), ukusebenza kahle kwe-PSII okuphezulu (Fv/Fm), i-photochemical maximum quantum yield ye-PSII (Fv/F0 ) kanye ne-Fm/F0 ezitshalweni zezinhlobo ezimbili zerayisi [Federrose 67 (F67) kanye ne-Federrose 2000 (F2000)] ezinsukwini ezingama-55 ngemva kokuvela (DAE)).
Okuqukethwe kwamanzi ahlobene (RWC) kwezitshalo zerayisi eziphathwe ngendlela ehlukile kubonise umehluko (P ≤ 0.05) ekusebenzisaneni phakathi kokwelashwa kokuhlola nokwelashwa kwamaqabunga (Isithombe 1A). Lapho kwelashwa nge-SA, amanani aphansi kakhulu aqoshwa kuzo zombili izinhlobo zezakhi zofuzo (74.01% ku-F67 kanye no-76.6% ku-F2000). Ngaphansi kwezimo zokucindezeleka kokushisa, i-RWC yezitshalo zerayisi zazo zombili izinhlobo zezakhi zofuzo eziphathwe ngama-phytohormone ahlukene yanda kakhulu. Sekukonke, ukusetshenziswa kwamaqabunga kwe-CK, GA, AUX, noma i-BR kwandise i-RWC yaya kumanani afana nalawo ezitshalo ezikhuliswe ngaphansi kwezimo ezinhle ngesikhathi sokuhlolwa. Ukulawula okuphelele kanye nezitshalo ezifuthwe ngama-foliar kuqophe amanani angaba ngu-83% kuzo zombili izinhlobo zezakhi zofuzo. Ngakolunye uhlangothi, ama-gs nawo abonise umehluko omkhulu (P ≤ 0.01) ekusebenzisaneni kokuhlolwa nokwelashwa (Isithombe 1B). Isitshalo esilawula ngokuphelele (AC) siphinde saqopha amanani aphezulu kakhulu kuhlobo ngalunye lwe-genotype (440.65 mmol m-2s-1 ye-F67 kanye ne-511.02 mmol m-2s-1 ye-F2000). Izitshalo zelayisi ezibhekene nokucindezeleka kokushisa okuhlangene zodwa zibonise amanani aphansi kakhulu e-gs kuzo zombili izinhlobo ze-genotype (150.60 mmol m-2s-1 ye-F67 kanye ne-171.32 mmol m-2s-1 ye-F2000). Ukwelashwa kwamaqabunga ngabo bonke abalawuli bokukhula kwezitshalo nakho kukhuphuke g. Ezitshalweni zelayisi ze-F2000 ezifuthwe nge-CC, umphumela wokufutha ngama-phytohormones wawusobala kakhulu. Leli qembu lezitshalo alibonisanga mehluko uma kuqhathaniswa nezitshalo ezilawula ngokuphelele (AC 511.02 kanye ne-CC 499.25 mmol m-2s-1).
Umfanekiso 1. Umphumela wokucindezeleka kokushisa okuhlangene (40°/30°C emini/ebusuku) kokuqukethwe kwamanzi okuhlobene (RWC) (A), ukuqhutshwa kwesisu (gs) (B), ukukhiqizwa kwe-malondialdehyde (MDA) (C), kanye nokuqukethwe kwe-proline. (D) ezitshalweni zezinhlobo ezimbili zerayisi (F67 kanye ne-F2000) ezinsukwini ezingama-55 ngemva kokuvela (DAE). Ukwelashwa okuhlolwe uhlobo ngalunye lwe-genotype kufakiwe: ukulawula okuphelele (AC), ukulawula ukucindezeleka kokushisa (SC), ukucindezeleka kokushisa + auxin (AUX), ukucindezeleka kokushisa + gibberellin (GA), ukucindezeleka kokushisa + i-cell mitogen (CK), kanye nokucindezeleka kokushisa + i-brassinosteroid. (BR). Ikholomu ngayinye imelela iphutha elijwayelekile ± lamaphoyinti edatha amahlanu (n = 5). Amakholomu alandelwa izinhlamvu ezahlukene abonisa umehluko obalulekile ngokwezibalo ngokusho kokuhlolwa kukaTukey (P ≤ 0.05). Izinhlamvu ezinesibonakaliso esilinganayo zibonisa ukuthi isilinganiso asibalulekile ngokwezibalo (≤ 0.05).
Okuqukethwe yi-MDA (P ≤ 0.01) kanye ne-proline (P ≤ 0.01) nakho kubonise umehluko omkhulu ekusebenzisaneni phakathi kokuhlolwa kanye nokwelashwa nge-phytohormone (Isithombe 1C, D). Ukwanda kwe-lipid peroxidation kwabonwa ngokwelashwa kwe-SC kuzo zombili izinhlobo ze-genotype (Isithombe 1C), noma kunjalo izitshalo eziphathwe nge-leaf growth regulator spray zibonise ukwehla kwe-lipid peroxidation kuzo zombili izinhlobo ze-genotype; Ngokuvamile, ukusetshenziswa kwama-phytohormones (CA, AUC, BR noma GA) kuholela ekunciphiseni kwe-lipid peroxidation (okuqukethwe yi-MDA). Akukho mehluko owatholakala phakathi kwezitshalo ze-AC zezinhlobo ezimbili ze-genotypes kanye nezitshalo ezingaphansi kokucindezeleka kokushisa futhi zafuthwa ngama-phytohormones (kubonwe amanani e-FW ezitshalweni ze-"F67" kusukela ku-4.38–6.77 µmol g-1, kanye nezitshalo ze-FW "F2000" "kubonwe amanani asukela ku-2.84 kuya ku-9.18 µmol g-1 (izitshalo). Ngakolunye uhlangothi, ukwenziwa kwe-proline ezitshalweni ze-"F67" kwakuphansi kunezitshalo ze-"F2000" ezingaphansi kokucindezeleka okuhlangene, okwaholela ekwandeni kokukhiqizwa kwe-proline. ezitshalweni zerayisi ezicindezelwe ukushisa, kuzo zombili izivivinyo, kwabonwa ukuthi ukuphathwa kwala ma-hormone kwandisa kakhulu okuqukethwe kwe-amino acid kwezitshalo ze-F2000 (i-AUX ne-BR kwakungu-30.44 no-18.34 µmol g-1) ngokulandelana (Isithombe 1G).
Imiphumela yesifutho sokulawula ukukhula kwezitshalo eziluhlaza kanye nokucindezeleka kokushisa okuhlangene ekushiseni kwezitshalo eziluhlaza kanye nenkomba yokubekezelelana okuhlobene (RTI) kuboniswe kuZithombe 2A no-B. Kuzo zombili izinhlobo ze-genotype, izinga lokushisa lezitshalo eziluhlaza eziluhlaza ezitshalweni ze-AC laliseduze no-27°C, kanti elezitshalo ze-SC lalicishe libe ngu-28°C. WITH. Kwaphinde kwabonwa ukuthi ukwelashwa kwezitshalo eziluhlaza nge-CK kanye ne-BR kwaholela ekwehleni kwezinga lokushisa lezitshalo eziluhlaza ngo-2–3°C uma kuqhathaniswa nezitshalo ze-SC (Isithombe 2A). I-RTI ibonise ukuziphatha okufanayo nezinye izinto eziphilayo, ikhombisa umehluko omkhulu (P ≤ 0.01) ekuxhumaneni phakathi kokuhlolwa kanye nokwelashwa (Isithombe 2B). Izitshalo ze-SC zibonise ukubekezelelana okuphansi kwezitshalo kuzo zombili izinhlobo ze-genotype (34.18% kanye no-33.52% wezitshalo zerayisi "F67" kanye "F2000", ngokulandelana). Ukudla kwezitshalo eziluhlaza ngamahlamvu kuthuthukisa i-RTI ezitshalweni ezivezwe ekucindezelekeni okuphezulu kokushisa. Lo mphumela ubonakale kakhulu ezitshalweni ze-“F2000” ezifuthwe nge-CC, lapho i-RTI yayingu-97.69. Ngakolunye uhlangothi, umehluko omkhulu wabonwa kuphela ku-index yokucindezeleka kwesivuno (CSI) yezitshalo zerayisi ngaphansi kwezimo zokucindezeleka kwe-foliar factor (P ≤ 0.01) (Isithombe 2B). Izitshalo zerayisi kuphela ezitholwe ekucindezelweni kokushisa okuyinkimbinkimbi ezibonise inani eliphakeme kakhulu le-index yokucindezeleka (0.816). Lapho izitshalo zerayisi zifuthwa ngama-phytohormone ahlukahlukene, i-index yokucindezeleka yayiphansi (amanani asuka ku-0.6 kuya ku-0.67). Ekugcineni, isitshalo serayisi esikhule ngaphansi kwezimo ezifanele sasinonani elingu-0.138.
Isithombe 2. Imiphumela yokucindezeleka kokushisa okuhlangene (40°/30°C emini/ebusuku) ekushiseni kwekhanophi (A), inkomba yokubekezelelana okuhlobene (RTI) (B), kanye nenkomba yokucindezeleka kwezitshalo (CSI) (C) yezinhlobo ezimbili zezitshalo. Izinhlobo zerayisi zezentengiselwano (F67 kanye ne-F2000) zenziwe ukwelashwa okuhlukene kokushisa. Ukwelashwa okuhlolwe uhlobo ngalunye lwe-genotype kufakiwe: ukulawula okuphelele (AC), ukulawula ukucindezeleka kokushisa (SC), ukucindezeleka kokushisa + i-auxin (AUX), ukucindezeleka kokushisa + i-gibberellin (GA), ukucindezeleka kokushisa + i-cell mitogen (CK), kanye nokucindezeleka kokushisa + i-brassinosteroid. (BR). Ukucindezeleka kokushisa okuhlangene kuhilela ukuveza izitshalo zerayisi emazingeni okushisa aphezulu emini/ebusuku (40°/30°C emini/ebusuku). Ikholomu ngayinye imelela iphutha elijwayelekile ± lamaphuzu edatha amahlanu (n = 5). Amakholomu alandelwa izinhlamvu ezahlukene abonisa umehluko obalulekile ngokwezibalo ngokusho kokuhlolwa kukaTukey (P ≤ 0.05). Izinhlamvu ezinesibonakaliso esilinganayo zibonisa ukuthi isilinganiso asibalulekile ngokwezibalo (≤ 0.05).
Ukuhlaziywa kwezingxenye eziyinhloko (i-PCA) kwembule ukuthi iziguquguquko ezihlolwe ku-55 DAE zichaze u-66.1% wezimpendulo zomzimba neze-biochemical zezitshalo zerayisi ezicindezelwe ukushisa eziphathwe nge-growth regulator spray (Isithombe 3). Ama-vector amelela iziguquguquko kanye namachashazi amelela abalawuli bokukhula kwezitshalo (ama-GR). Ama-vector e-gs, okuqukethwe kwe-chlorophyll, ukusebenza kahle kwe-quantum okuphezulu kwe-PSII (Fv/Fm) kanye namapharamitha e-biochemical (TChl, MDA kanye ne-proline) aseduze nomsuka, okubonisa ubudlelwano obuphezulu phakathi kokuziphatha komzimba kwezitshalo kanye nazo. eziguquguqukayo. Iqembu elilodwa (V) lifaka izithombo zerayisi ezikhuliswe ekushiseni okuphezulu (AT) kanye nezitshalo ze-F2000 eziphathwe nge-CK kanye ne-BA. Ngesikhathi esifanayo, iningi lezitshalo eziphathwe nge-GR zakha iqembu elihlukile (IV), kanti ukwelashwa nge-GA ku-F2000 kwakha iqembu elihlukile (II). Ngokuphambene nalokho, izithombo zerayisi ezicindezelwe ukushisa (amaqembu I no-III) ngaphandle kokufuthwa kwama-phytohormones amaqabunga (zombili izinhlobo ze-genotypes zaziyi-SC) zatholakala endaweni ephambene neqembu V, okubonisa umphumela wokucindezeleka kokushisa emzimbeni wezitshalo.
Umfanekiso 3. Ukuhlaziywa kwe-bigraphical yemiphumela yokucindezeleka kokushisa okuhlangene (40°/30°C imini/ubusuku) ezitshalweni zezinhlobo ezimbili zerayisi (F67 kanye ne-F2000) ezinsukwini ezingama-55 ngemva kokuvela (DAE). Izifinyezo: AC F67, ukulawula okuphelele F67; SC F67, ukulawula ukucindezeleka kokushisa F67; AUX F67, ukucindezeleka kokushisa + auxin F67; GA F67, ukucindezeleka kokushisa + gibberellin F67; CK F67, ukucindezeleka kokushisa + ukuhlukaniswa kwamaseli BR F67, ukucindezeleka kokushisa + i-brassinosteroid. F67; AC F2000, ukulawula okuphelele F2000; SC F2000, Ukulawula Ukucindezeleka Kokushisa F2000; AUX F2000, ukucindezeleka kokushisa + auxin F2000; GA F2000, ukucindezeleka kokushisa + gibberellin F2000; I-CK F2000, ukucindezeleka kokushisa + i-cytokinin, i-BR F2000, ukucindezeleka kokushisa + i-steroid yethusi; F2000.
Izinto eziguquguqukayo ezifana nokuqukethwe kwe-chlorophyll, ukuhanjiswa kwesisu, isilinganiso se-Fv/Fm, i-CSI, i-MDA, i-RTI kanye nokuqukethwe kwe-proline kungasiza ekuqondeni ukuguquguquka kwezinhlobo zerayisi nokuhlola umthelela wamasu ezolimo ngaphansi kokucindezeleka kokushisa (Sarsu et al., 2018; Quintero-Calderon et al., 2021). Inhloso yalolu vivinyo kwakuwukuhlola umphumela wokusetshenziswa kwezilawuli ezine zokukhula kumapharamitha okuphila kanye namakhemikhali ezithombo zerayisi ngaphansi kwezimo eziyinkimbinkimbi zokucindezeleka kokushisa. Ukuhlolwa kwezithombo kuyindlela elula futhi esheshayo yokuhlola ngesikhathi esisodwa izitshalo zerayisi kuye ngobukhulu noma isimo sengqalasizinda etholakalayo (Sarsu et al. 2018). Imiphumela yalolu cwaningo ikhombisile ukuthi ukucindezeleka kokushisa okuhlangene kubangela izimpendulo ezahlukene zomzimba kanye namakhemikhali kuzinhlobo ezimbili zerayisi, okubonisa inqubo yokuguquguquka. Le miphumela ikhombisa nokuthi izifutho zokulawula ukukhula kwamaqabunga (ikakhulukazi ama-cytokinins nama-brassinosteroids) zisiza ilayisi ukuthi livumelane nokucindezeleka kokushisa okuyinkimbinkimbi njengoba i-favor ithinta kakhulu ama-gs, i-RWC, isilinganiso se-Fv/Fm, ama-pigment e-photosynthetic kanye nokuqukethwe kwe-proline.
Ukusetshenziswa kwezilawuli zokukhula kusiza ukuthuthukisa isimo samanzi ezitshalo zelayisi ngaphansi kokucindezeleka kokushisa, okungase kuhlotshaniswe nokucindezeleka okuphezulu kanye namazinga okushisa aphansi ezitshalo. Lolu cwaningo lubonise ukuthi phakathi kwezitshalo "ze-F2000" (i-genotype ethintekayo), izitshalo zelayisi eziphathwe ngokuyinhloko nge-CK noma i-BR zazinamanani aphezulu e-gs kanye namanani aphansi e-PCT kunezitshalo eziphathwe nge-SC. Izifundo zangaphambilini zibonise nokuthi i-gs kanye ne-PCT ziyizinkomba ezinembile zomzimba ezinganquma impendulo eguquguqukayo yezitshalo zelayisi kanye nemiphumela yamasu ezolimo ekucindezelweni kokushisa (Restrepo-Diaz noGarces-Varon, 2013; Sarsu et al., 2018; Quintero). -Carr DeLong et al., 2021). I-Leaf CK noma i-BR ithuthukisa i-g ngaphansi kokucindezeleka ngoba la ma-hormone ezitshalo angakhuthaza ukuvulwa kwe-stomatal ngokusebenzisana kokwenziwa namanye ama-molecule esignaling njenge-ABA (umgqugquzeli wokuvalwa kwe-stomatal ngaphansi kokucindezeleka kwe-abiotic) (Macková et al., 2013; Zhou et al., 2013). 2013). ). , 2014). Ukuvulwa kwesisu kukhuthaza ukupholisa amaqabunga futhi kusiza ukunciphisa amazinga okushisa e-canopy (Sonjaroon et al., 2018; Quintero-Calderón et al., 2021). Ngenxa yalezi zizathu, izinga lokushisa le-canopy lezitshalo zerayisi ezifuthwe nge-CK noma i-BR lingase libe phansi ngaphansi kokucindezeleka kokushisa okuhlangene.
Ukucindezeleka okuphezulu kokushisa kunganciphisa okuqukethwe kwe-pigment ye-photosynthesis yamaqabunga (Chen et al., 2017; Ahammed et al., 2018). Kulolu cwaningo, lapho izitshalo zerayisi zazingaphansi kokucindezeleka kokushisa futhi zingafuthwa nganoma yiziphi izilawuli zokukhula kwezitshalo, imibala ye-photosynthesis yayivame ukwehla kuzo zombili izinhlobo ze-genophyl (Ithebula 2). UFeng et al. (2013) nabo babike ukwehla okukhulu kokuqukethwe kwe-chlorophyll emaqabunga ezinhlobo ezimbili ze-genophyl zikakolweni ezivezwe ekucindezelekeni kokushisa. Ukuchayeka emazingeni okushisa aphezulu kuvame ukuholela ekwehleni kokuqukethwe kwe-chlorophyll, okungase kube ngenxa yokwehla kwe-biosynthesis ye-chlorophyll, ukuwohloka kwemibala, noma imiphumela yazo ehlangene ngaphansi kokucindezeleka kokushisa (Fahad et al., 2017). Kodwa-ke, izitshalo zerayisi ezelashwe kakhulu nge-CK kanye ne-BA zandise ukuhlushwa kwemibala ye-photosynthesis yamaqabunga ngaphansi kokucindezeleka kokushisa. Imiphumela efanayo nayo yabikwa nguJespersen noHuang (2015) kanye noSuchsagunpanit et al. (2015), owabona ukwanda kokuqukethwe kwe-chlorophyll yamaqabunga ngemuva kokusetshenziswa kwama-hormone e-zeatin ne-epibrassinosteroid ku-bentgrass ecindezelwe ukushisa kanye nelayisi, ngokulandelana. Incazelo enengqondo yokuthi kungani i-CK ne-BR zikhuthaza ukwanda kokuqukethwe kwe-chlorophyll yamaqabunga ngaphansi kokucindezeleka kokushisa okuhlangene ukuthi i-CK ingathuthukisa ukuqaliswa kokungeniswa okuqhubekayo kwabagqugquzeli bokubonakaliswa (njenge-promoter evuselelayo yokukhula (SAG12) noma i-promoter ye-HSP18) futhi inciphise ukulahleka kwe-chlorophyll emaqabunga. , ukubambezeleka kokukhula kweqabunga futhi kwandise ukumelana nezitshalo ekushiseni (Liu et al., 2020). I-BR ingavikela i-chlorophyll yamaqabunga futhi yandise okuqukethwe kwe-chlorophyll yamaqabunga ngokusebenzisa noma ngokubangela ukwakheka kwama-enzyme ahilelekile ku-biosynthesis ye-chlorophyll ngaphansi kwezimo zokucindezeleka (Sharma et al., 2017; Siddiqui et al., 2018). Okokugcina, ama-phytohormone amabili (i-CK ne-BR) nawo akhuthaza ukuvezwa kwamaprotheni okushisa futhi athuthukise izinqubo ezahlukahlukene zokuzivumelanisa nezimo, njengokwanda kwe-chlorophyll biosynthesis (Sharma et al., 2017; Liu et al., 2020).
I-Chlorophyll, ipharamitha ye-fluorescence, inikeza indlela esheshayo nengabhubhisi engahlola ukubekezelelana kwezitshalo noma ukuzivumelanisa nezimo zokucindezeleka okungena-abiotic (Chaerle et al. 2007; Kalaji et al. 2017). Amapharamitha afana nesilinganiso se-Fv/Fm asetshenziswe njengezinkomba zokuzivumelanisa nezitshalo nezimo zokucindezeleka (Alvarado-Sanabria et al. 2017; Chavez-Arias et al. 2020). Kulesi sifundo, izitshalo ze-SC zibonise amanani aphansi kakhulu alesi siguquguquki, ikakhulukazi izitshalo zerayisi "ze-F2000". U-Yin et al. (2010) bathole nokuthi isilinganiso se-Fv/Fm samaqabunga erayisi aphezulu kakhulu sehle kakhulu emazingeni okushisa angaphezu kuka-35°C. Ngokusho kukaFeng et al. (2013), isilinganiso se-Fv/Fm esiphansi ngaphansi kokucindezeleka kokushisa sibonisa ukuthi izinga lokubamba amandla okuvuselela nokuguqulwa yisikhungo sokusabela se-PSII liyancipha, okubonisa ukuthi isikhungo sokusabela se-PSII siyaqhekeka ngaphansi kokucindezeleka kokushisa. Lokhu kuqaphela kusenza siphethe ngokuthi ukuphazamiseka emshinini we-photosynthetic kubonakala kakhulu ezinhlotsheni ezizwelayo (Fedearroz 2000) kunasezinhlotsheni ezimelana nezifo (Fedearroz 67).
Ukusetshenziswa kwe-CK noma i-BR ngokuvamile kuthuthukise ukusebenza kwe-PSII ngaphansi kwezimo eziyinkimbinkimbi zokucindezeleka kokushisa. Imiphumela efanayo itholwe nguSuchsagunpanit et al. (2015), abaqaphele ukuthi ukusetshenziswa kwe-BR kwandisa ukusebenza kahle kwe-PSII ngaphansi kokucindezeleka kokushisa elayisini. UKumar et al. (2020) bathole nokuthi izitshalo ze-chickpea eziphathwe nge-CK (6-benzyladine) futhi ezicindezelwe ukushisa kwandisa isilinganiso se-Fv/Fm, baphetha ngokuthi ukusetshenziswa kwe-CK ngamahlamvu ngokusebenzisa umjikelezo we-zeaxanthin pigment kukhuthaza umsebenzi we-PSII. Ngaphezu kwalokho, i-BR leaf spray ithanda i-PSII photosynthesis ngaphansi kwezimo ezihlanganisiwe zokucindezeleka, okubonisa ukuthi ukusetshenziswa kwale phytohormone kuholele ekunciphiseni ukusabalala kwamandla okuvuselela ama-antennae e-PSII futhi kwakhuthaza ukuqongelela kwamaprotheni amancane okushisa kuma-chloroplast (Ogweno et al. 2008; Kothari noLachowitz). , 2021).
Okuqukethwe yi-MDA kanye ne-proline kuvame ukwanda lapho izitshalo zingaphansi kokucindezeleka okungenabungozi uma kuqhathaniswa nezitshalo ezikhuliswe ngaphansi kwezimo ezinhle (Alvarado-Sanabria et al. 2017). Izifundo zangaphambilini ziphinde zabonisa ukuthi amazinga e-MDA kanye ne-proline ayizinkomba ze-biochemical ezingasetshenziswa ukuqonda inqubo yokuzivumelanisa nezimo noma umthelela wemikhuba yezolimo elayisini ngaphansi kwamazinga okushisa aphezulu emini noma ebusuku (Alvarado-Sanabria et al., 2017; Quintero-Calderón et al. . , 2021). Lezi zifundo ziphinde zabonisa ukuthi okuqukethwe yi-MDA kanye ne-proline kuvame ukuba phezulu ezitshalweni zerayisi ezivezwe emazingeni okushisa aphezulu ebusuku noma emini, ngokulandelana. Kodwa-ke, ukufafaza nge-CK kanye ne-BR kwafaka isandla ekwehleni kwe-MDA kanye nokwenyuka kwamazinga e-proline, ikakhulukazi ku-genotype ebekezelayo (Federroz 67). I-CK spray ingakhuthaza ukuvezwa ngokweqile kwe-cytokinin oxidase/dehydrogenase, ngaleyo ndlela kwandise okuqukethwe kwamakhemikhali avikelayo njenge-betaine kanye ne-proline (Liu et al., 2020). I-BR ikhuthaza ukuqaliswa kwama-osmoprotectants afana ne-betaine, ushukela, nama-amino acid (kufaka phakathi i-proline yamahhala), igcina ibhalansi ye-osmotic yeselula ngaphansi kwezimo eziningi zemvelo ezimbi (Kothari noLachowiec, 2021).
Inkomba yokucindezeleka kwezitshalo (i-CSI) kanye nenkomba yokubekezelelana okuhlobene (i-RTI) zisetshenziselwa ukunquma ukuthi ukwelashwa okuhlolwayo kuyasiza yini ekunciphiseni ukucindezeleka okuhlukahlukene (okungamakhemikhali kanye nokuphilayo) futhi kube nomthelela omuhle emzimbeni wezitshalo (uCastro-Duque et al., 2020; uChavez-Arias et al., 2020). Amanani e-CSI angasukela ku-0 kuya ku-1, amele izimo ezingezona ezokucindezeleka kanye nokucindezeleka, ngokulandelana (uLee et al., 2010). Amanani e-CSI ezitshalo ezicindezelwe ukushisa (i-SC) asukela ku-0.8 kuya ku-0.9 (Isithombe 2B), okubonisa ukuthi izitshalo zerayisi zithinteke kabi ukucindezeleka okuhlangene. Kodwa-ke, ukufafaza i-BC (0.6) noma i-CK (0.6) ngamahlamvu kuholele kakhulu ekwehleni kwalesi sibonakaliso ngaphansi kwezimo zokucindezeleka okungamakhemikhali uma kuqhathaniswa nezitshalo zerayisi ze-SC. Ezitshalweni ze-F2000, i-RTI ibonise ukwanda okuphezulu lapho kusetshenziswa i-CA (97.69%) kanye ne-BC (60.73%) uma kuqhathaniswa ne-SA (33.52%), okubonisa ukuthi laba balawuli bokukhula kwezitshalo nabo banegalelo ekuthuthukiseni impendulo yelayisi ekubekezeleni kokwakheka. Ukushisa ngokweqile. Lezi zinkomba ziye zaphakanyiswa ukuphatha izimo zokucindezeleka ezinhlotsheni ezahlukene. Ucwaningo olwenziwe nguLee et al. (2010) lubonise ukuthi i-CSI yezinhlobo ezimbili zikakotini ngaphansi kokucindezeleka kwamanzi okumaphakathi yayicishe ibe ngu-0.85, kanti amanani e-CSI ezinhlobo eziniselwa kahle ayesukela ku-0.4 kuya ku-0.6, ephetha ngokuthi le nkomba iyinkomba yokuguquguquka kwamanzi kwezimo zokucindezeleka. Ngaphezu kwalokho, uChavez-Arias et al. (2020) bahlole ukusebenza kahle kwe-encitors zokwenziwa njengesu eliphelele lokuphatha ukucindezeleka ezitshalweni ze-C. elegans futhi bathola ukuthi izitshalo ezifuthwe ngalezi zinhlanganisela zibonise i-RTI ephezulu (65%). Ngokusekelwe kulokhu okungenhla, i-CK kanye ne-BR zingabhekwa njengezindlela zezolimo ezihlose ukwandisa ukubekezelelana kwelayisi ekucindezelweni kokushisa okuyinkimbinkimbi, njengoba laba balawuli bokukhula kwezitshalo bekhuthaza izimpendulo ezinhle zamakhemikhali kanye nezomzimba.
Eminyakeni embalwa edlule, ucwaningo lwelayisi eColombia lugxile ekuhloleni izinhlobo zezakhi zofuzo ezibekezelela amazinga okushisa aphezulu emini noma ebusuku kusetshenziswa izici zomzimba noma ze-biochemical (Sánchez-Reinoso et al., 2014; Alvarado-Sanabria et al., 2021). Kodwa-ke, eminyakeni embalwa edlule, ukuhlaziywa kobuchwepheshe obusebenzayo, obunomnotho nobunenzuzo kuye kwaba kubaluleke kakhulu ukuphakamisa ukuphathwa kwezitshalo okuhlanganisiwe ukuze kuthuthukiswe imiphumela yezikhathi eziyinkimbinkimbi zokucindezeleka kokushisa ezweni (Calderón-Páez et al., 2021; Quintero-Calderon et al., 2021). Ngakho-ke, izimpendulo zomzimba neze-biochemical zezitshalo zerayisi ekucindezelweni kokushisa okuyinkimbinkimbi (40°C ngosuku/30°C ebusuku) ezibonwe kulolu cwaningo ziphakamisa ukuthi ukufafaza ngamahlamvu nge-CK noma i-BR kungaba yindlela efanele yokuphatha izitshalo ukunciphisa imiphumela emibi. Umphumela wezikhathi zokucindezeleka kokushisa okumaphakathi. Lokhu kwelashwa kuthuthukise ukubekezelelana kwezinhlobo zezakhi zofuzo zerayisi (i-CSI ephansi kanye ne-RTI ephezulu), okubonisa ukuthambekela okujwayelekile ezimpendulweni zomzimba neze-biochemical zezitshalo ngaphansi kokucindezeleka kokushisa okuhlangene. Impendulo eyinhloko yezitshalo zerayisi kwaba ukwehla kokuqukethwe kwe-GC, i-chlorophyll iyonke, ama-chlorophyll α kanye ne-β kanye nama-carotenoids. Ngaphezu kwalokho, izitshalo zihlushwa umonakalo we-PSII (imingcele ye-fluorescence ye-chlorophyll enciphile njengesilinganiso se-Fv/Fm) kanye nokwanda kwe-lipid peroxidation. Ngakolunye uhlangothi, lapho irayisi iphathwa nge-CK kanye ne-BR, le miphumela emibi yancishiswa futhi okuqukethwe kwe-proline kwanda (Isithombe 4).
Isithombe 4. Imodeli yomqondo yemiphumela yokucindezeleka kokushisa okuhlangene kanye nesifutho sokulawula ukukhula kwezitshalo ezitshalweni zerayisi. Imicibisholo ebomvu neluhlaza okwesibhakabhaka ikhombisa imiphumela emibi noma emihle yokusebenzisana phakathi kokucindezeleka kokushisa kanye nokusetshenziswa kwe-BR (brassinosteroid) kanye ne-CK (cytokinin) ezimpendulweni zomzimba neze-biochemical, ngokulandelana. gs: ukuqhutshwa kwesisu; Ingqikithi ye-Chl: okuqukethwe kwe-chlorophyll okuphelele; I-Chl α: okuqukethwe kwe-chlorophyll β; I-Cx+c: okuqukethwe kwe-carotenoid;
Ngamafuphi, izimpendulo zomzimba kanye nezamakhemikhali kulolu cwaningo zibonisa ukuthi izitshalo zerayisi ze-Fedearroz 2000 zisengozini enkulu yesikhathi sokucindezeleka okushisa okuyinkimbinkimbi kunezitshalo zerayisi ze-Fedearroz 67. Bonke abalawuli bokukhula abahlolwe kulolu cwaningo (ama-auxins, ama-gibberellins, ama-cytokinins, noma ama-brassinosteroids) babonise izinga elithile lokunciphisa ukucindezeleka kokushisa okuhlangene. Kodwa-ke, ama-cytokinin nama-brassinosteroids abangele ukuzivumelanisa kangcono nezitshalo njengoba bobabili abalawuli bokukhula kwezitshalo bandise okuqukethwe kwe-chlorophyll, imingcele ye-alpha-chlorophyll fluorescence, ama-gs kanye ne-RWC uma kuqhathaniswa nezitshalo zerayisi ngaphandle kokusetshenziswa, futhi kwanciphisa okuqukethwe kwe-MDA kanye nokushisa kwekhanophi. Ngamafuphi, siphetha ngokuthi ukusetshenziswa kwabalawuli bokukhula kwezitshalo (ama-cytokinins nama-brassinosteroids) kuyithuluzi eliwusizo ekuphatheni izimo zokucindezeleka ezitshalweni zerayisi ezibangelwa ukucindezeleka okukhulu kokushisa ngezikhathi zokushisa okuphezulu.
Izinto zokuqala ezethulwe ocwaningweni zifakiwe kulesi sihloko, futhi imibuzo eyengeziwe ingaqondiswa kumbhali ohambisanayo.