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Objašnjenje zašto više iona Ca2+ uzrokuje dulju duljinu korijena u biljkama

Objašnjenje zašto više iona Ca2+ uzrokuje dulju duljinu korijena u biljkama



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Ovo je za eksperiment radi provjere dostupnosti mineralnih iona u biljkama. Dakle, ako u tlu ima više kalcijevih iona, došlo bi do povećanja duljine korijena biljke na kojoj se vrši ispitivanje. Zašto bi to bio slučaj? Kalcijevi ioni čine kalcijev pektat u srednjoj lameli i uzrokuju da stanična stijenka postane krutija i čvršća. Ali zašto bi to uzrokovalo povećanje duljine korijena?


Omjer korijena i pucanja

5.12.1 Arhitektonske značajke postrojenja povezane s učinkovitošću uporabe fosfora

Za optimalno usvajanje fosfora osobine korijena očito su važne. Nedostatak fosfora obično dovodi do većeg omjera korijena: izdanaka i do promjena u arhitekturi korijena. Važan doprinos uklanjanju tla iz fosfora je proširenje korijena, jer se rizosfera brzo osiromaši fosforom, a osipanjem fosfora ovim područjem difuzijom i mobilizacijom obično se ne ide u korak s usisavanjem. Štoviše, fosfor često nije ravnomjerno raspoređen u tlu. Nekoliko arhitektonskih promjena povezano je s većom učinkovitošću korištenja fosfora iz korijena. Veći broj bočnih korijena dovodi do poboljšanih mogućnosti uklanjanja fosfora (Lynch, 2007). Nekoliko svojstava smanjit će troškove ugljika za proizvodnju ovih bočnih korijena, uključujući tanju i produženiju morfologiju i anatomske značajke, poput manjeg sekundarnog rasta stele i aerenhima u korteksu (Zhu i sur., 2010.). Povećano bočno grananje korijena obično je popraćeno smanjenjem primarnog razvoja korijena (Niu i sur., 2013b). Povećanje aksijalne duljine korijena, bez povećanog bočnog grananja, također je pronađeno u kukuruzu i običnom grahu, te je protumačeno kao istraživačko ponašanje za zakrpe tla obogaćene fosforom gdje bi bočno formiranje korijena tada postalo funkcionalno (Richardson et al., 2011 ). Promjena rasta korijena može se postići uz relativno niske troškove ugljika. Povećane gustoće i duljine dovode do povećanja kapaciteta apsorpcije fosfora (Wang i sur., 2004. Yan i sur., 2004.).

Budući da je gornji dio tla često bogatiji fosforom, prilagodbe strukture korijena koje povećavaju gustoću korijena u gornjim dijelovima tla pogoduju učinkovitosti usvajanja fosfora. To se postiže plićim kutom rasta aksijalnih korijena, što dovodi do veće duljine korijena u vrhu tla. Mahunarke obični grah i soja mogu se odabrati za veći broj zrna bazalnog korijena, to jest bočne korijene koji se pojavljuju u "prstenovima" na prijelaznoj zoni između mladice i korijena (Lynch, 2007). Osim toga, Wang i sur. (2004) pokazali su u soji da su gustoća korijena i ukupna duljina veće u bazalnom korijenu od korijena slavine, dok je koncentracija biljnog fosfora pozitivno povezana s gustoćom korijena dlake. Također su pokazali negativnu korelaciju između gustoće korijena i prosječne duljine korijena, što bi se moglo protumačiti kao kompromis u smislu učinkovitosti korištenja ugljika, odnosno kombiniranje velike gustoće i duljine vlasi korijena bilo bi preskupo u uvjeti unosa ugljika. S druge strane, Yan i sur. (2004) pronašli su i gušće i duže dlačice korijena u fosforno učinkovitim genotipovima običnog graha. Očigledno nemaju svi usjevi sposobnost povećanja stvaranja dlaka na korijenu radi povećanja sposobnosti usvajanja fosfora. Na primjer, luku uglavnom nedostaje ovo svojstvo (Liu i James, 2006). Prema Ochoa i sur. (2006), kod nekih usjeva bit će korisno poboljšati nastanak korijena. Međutim, povećano privremeno stvaranje korijena može doći po cijenu manje bočnog ukorjenjivanja (Walk et al., 2006). Promjene u gustoći i kutu rasta bočnih korijena također povećavaju kapacitet čišćenja u gornjem sloju tla (Ao i sur., 2010.).

Poseban slučaj su proteoidni korijeni (korijeni grozdova: gusti grozdovi kratkih bočnih korijena) pronađeni pri niskom sadržaju fosfora u lupini i divljim vrstama Proteaceae obitelji (Shane i Lambers, 2005. Lambers i sur., 2011., 2013.). Oni su također najistaknutiji u gornjem sloju tla i smatra se da su uglavnom učinkoviti mobiliziranjem fosfora izlučivanjem organskih kiselina, a ne pročišćavanjem kroz proširenje korijena (Rath i sur., 2010.). Lupini kombiniraju ovu osobinu s odsustvom mikorize. Arhitektonske značajke korijena, poput bočnog grananja i gustoće vlasi korijena, očito su povoljne za učinkovitost upotrebe fosfora. Međutim, praćenje ovih osobina i njihovo korištenje za odabir u uzgojnim programima zasigurno nije jednostavno.


MOBILNOST I UZIMANJE KALCIJA BILJKAMA

Unos kalcija u biljku je pasivan i ne zahtijeva unos energije. Mobilnost kalcija u biljci odvija se uglavnom u ksilemu, zajedno s vodom. Stoga je unos kalcija izravno povezan sa brzinom transpiracije biljke.

Uvjeti visoke vlažnosti, hladnoće i niske transpiracije mogu rezultirati nedostatkom kalcija. Nakupljanje slanosti također može uzrokovati nedostatak kalcija jer smanjuje unos vode u biljku.

Budući da je mobilnost kalcija u biljkama ograničena, nedostatak kalcija pojavit će se u mlađem lišću (odumrijeti ili opekline) i u plodovima (trulež kraja cvjetanja, gorka koštica), jer imaju vrlo nisku stopu transpiracije. Stoga je za stalni rast potrebno imati stalnu opskrbu kalcijem.


Tolerancija kiselog tla u pšenici

Brett F. Carver, James D. Ownby, in Advances in Agronomy, 1995

Uzročni elementi kiselosti tla

Kiselost tla određena je količinom aktivnosti H + u otopini tla, a na nju utječu edafski, klimatski i biološki čimbenici prirodne pojave (Johnson, 1988). Na primjer, tla koja se razvijaju od granitnih matičnih materijala zakiseljavaju brže od tla dobivenih od vapnenačkih matičnih materijala. Pješčana tla s relativno malo čestica gline brže se zakiseljuju zbog manjeg spremnika alkalnih kationa (puferski kapacitet) i većeg potencijala ispiranja. Prekomjerne količine oborina utječu na brzinu zakiseljavanja tla, ovisno o brzini cijeđenja vode kroz profil tla. Organska tvar će se raspasti i stvoriti ugljičnu kiselinu i druge slabe organske kiseline. Kumulativni učinak ovih čimbenika praktički je nemjerljiv tijekom nekoliko godina i stoga može relativno malo pridonijeti ukupnoj kiselosti tla.

Kiselost tla ne pokazuje samo kronološke varijacije, već i prostorne (vodoravne i okomite) varijacije. Tla izrazito dotrajala, poput brazilskih oksisola, kisela su po cijelom profilu. Nasuprot tome, Mollisols uzgojen kontinuiranom ozimom pšenicom u Oklahomi razvija fitotoksične razine kiselosti ograničene na površinski sloj (Tablica I). Tlo je u ovom primjeru klasificirano kao ilovača ilovače Pond Creek (Fino-muljevita, mješovita, mesična Pachic Argiustoll). Primao je godišnje primjene bezvodnog amonijaka od 1982. godine i vapnen je 6 godina prije uzorkovanja. Tipično za kisela tla pšeničnog pojasa Oklahome, površinske (0–30 cm) pH vrijednosti bile su kritično kisele, ali su podzemne (≥ 30 cm) srednje pH vrijednosti prešle 6,5. Kiselost tla također je pronađena u gornjih 30 cm za tla pšenice u južnom Novom Južnom Walesu (Fisher i Scott, 1987.).

Tablica I. Okomita raspodjela pH u polju pšenice u Oklahomi (3,0 hektara) Kontinuirano usjevano sa ozimom pšenicom i uzorkovano u mrežnom uzorku na dubinu od 1,2 m

Dubina (cm)pH aStandardna devijacija
MinimumMinimumSrednje
0-154.16.05.00.4
15-304.46.45.50.4
30-455.67.06.50.2
45-606.27.46.90.2
60-906.57.67.20.2
90-1206.27.77.20.3

Kiselost tla često se ubrzava u površinskom sloju određenim postupcima usjeva, npr. Ponovljenom primjenom dušika u višku usjeva usjeva. Neto proizvodnja H + nastaje prirodnim procesima, uključujući nitrifikaciju amonijačnog N:

Dio ove kiselosti neutralizira NO3 - preuzimanje i naknadno oslobađanje OH -. Drugi kompromitirajući čimbenici su denitrifikacija NO3 -, NH3 isparavanje ili NH4 + usvajanje od strane biljke. Prakse upravljanja koje optimiziraju učinkovitost upotrebe N i na kraju smanjuju količinu NO3 - izgubljeni ispiranjem mogli bi usporiti brzinu zakiseljavanja (Robson, 1989.).

Zakiseljavanje tla u površinskom sloju također se ubrzava uklanjanjem osnovnih kationa (Ca, Mg, K i Na) u ubranom proizvodu. Različite prakse usjeva pšenice povećavaju ili smanjuju potencijalnu kiselost uzrokovanu nitrifikacijom primijenjenog N, ovisno o relativnim količinama uklonjene stočne hrane, žitarica i/ili slame (a time i osnovnih kationa). Samo uklanjanje slame iscrpljuje osnovne katione u najvećoj mjeri i zapravo poboljšava zakiseljavanje nitrifikacijom (Westerman, 1987). Osiromašenje baze manje je značajno pri uklanjanju stočne hrane (ispašom ili sijenom), a najmanje pri uklanjanju zrna. Većina višenamjenskih sustava usjeva (npr. Uklanjanje krme i zrna) smanjuje potencijalnu kiselost uzrokovanu primjenom amonijačnog N, iako u različitim stupnjevima, ovisno o omjeru viška baze/N u različitim dijelovima biljke. Stoga će se razviti kiselost tla, a pH tla će se smanjivati, različitim brzinama, ovisno o sustavu usjeva. Ozbiljnost kiselosti tla raste s povećanjem prinosa vegetativne ili suhe tvari zrna. To može objasniti zašto je kiselost tla dosegla neviđene razine u nekim intenzivno upravljanim tlima Oklahome i Kansasa.


Transportne proteinske obitelji uključene u K + prehranu

K + -izborni kanali

Prvi transporter K + s ulogom u unosu hranjivih tvari bio je Shaker-nalik, naponski ograničeni i K + -selektivni kanal AKT1 (Hirsch et ਊl., 1998). Iako su naponski usmjereni (VG) kanali biljaka filogenetski povezani sa životinjskim Shaker kanala, oni su različiti i uključuju dodatne funkcionalne domene (Jegla et ਊl., 2018). Osnovna arhitektura VG kanala sastoji se od četiri podjedinice α koje okružuju središnju vodenu pora za prodiranje K +. Svaka podjedinica sadrži šest transmembranskih segmenata, nazvanih S1 –S6, koji se mogu podijeliti u dva različita modula: prve četiri α-spirale tvore domenu osjetnika napona koja sadrži više pozitivno nabijenih ostataka koji se kreću unutar membrane kao odgovor na napon . Ovaj pokret je izravno povezan s otvaranjem ili zatvaranjem kanala. Segmenti S5, S6 i petlja pora tvore domenu pora, nazvanu P, gdje svaka od četiri podjedinice jednako doprinosi putu prožimanja. Štoviše, biljne α-podjedinice imaju dugo C-terminalno područje koje čini više od polovice proteina (slika 2). Ovaj citosolni rep uključuje nekoliko funkcionalnih domena: (1) područje povezivanja (C-povezivač) proksimalno od pora koje transducira konformacijske promjene koje zatvaraju kanal i koje također mogu odrediti ciljnu membranu (Nieves-Cordones et ਊl., 2014b Jegla et ਊl., 2018.) (2) konzervirana i esencijalna homološka domena vezanja cikličkih nukleotida (CNBHD) čija funkcija nije vezanje cNMP-a već posreduje u interakcijama između podjedinica unutar tetramera kanala (3) ankirinske domene (pronađeno u samo šest od devet Arabidopsis VG kanali), koji mogu posredovati u vezanju međusobno povezanih proteina (Michaely i Bennett, 1992.) i (4) distalnu KT/KHA domenu bogatu hidrofobnim i kiselim ostacima, koja je jedinstvena za biljne K + kanale i uključena je u kanal tetramerizacija i grupiranje na membrani (Daram et ਊl., 1997 Ehrhardt et ਊl., 1997 Zimmermann et ਊl., 2001 Dreyer et ਊl., 2004).

Slika 2. Topološki modeli glavnih transportera iona uključenih u prehranu K +. (A) Napon K + kanali sadrže šest transmembranskih domena (S1 –S6) S4 je senzor napona koji karakterizira niz pozitivno nabijenih aminokiselina ( +). Dugi C-terminalni rep sadrži nekoliko očuvanih domena: C-povezivač, homolognu domenu koja se veže za cikličke nukleotide (CNBHD), ankirinsku domenu (ANK) i konačno područje bogato hidrofobnim i kiselim ostacima (KHA). (B) HKT transporteri imaju strukturu nalik kanalu koja sadrži četiri identične podjedinice (a 𠄽), a svaka se sastoji od dvije transmembranske spirale (M1 i M2) povezane P-petljom uključene u selektivnost iona. (C) KT/HAK/KUP transporteri imaju 12 pretpostavljenih transmembranskih domena (TM). Predviđeno je da se TM1-5 i TM6-10 presavijaju u istoj konformaciji, ali pokazuju obrnutu simetriju.

K + kanali s naponom pod nadzorom postrojenja podijeljeni su u tri potporodice s obzirom na njihov odgovor na membranski potencijal (Dreyer i Uozumi, 2011): (1) Unutarnji ispravljajući (Kin) kanali koji u Arabidopsis uključuju AKT1, AKT6, KAT1 i KAT2 koje otvaraju pri hiperpolariziranim membranskim potencijalima dopuštajući unos K +. (2) Vanjski ispravljajući (Kout) kanali koji posreduju oslobađanje K + jer se otvaraju pri depolariziranim membranskim potencijalima ovu skupinu čine SKOR i GORK kanali. (3) Slabo ispravljajući (Kweak) kanali koji mogu posredovati u preuzimanju i oslobađanju K +, a čiji Arabidopsis predstavnik je AKT2. Osim toga, Arabidopsis KC1 (KAT3) je električno nečujan Shaker-sličan protein koji stupa u interakciju i regulira funkcionalnost Kinovih kanala AKT1, KAT1, KAT2 i AKT2, ali ne i Kout kanale (Jeanguenin et ਊl., 2011). Ova interakcija negativno pomiče prag aktivacije Kinovih kanala i smanjuje makroskopsku unutarnju vodljivost u usporedbi s homomernim kanalima (Dreyer et ਊl., 1997. Duby et ਊl., 2008. Geiger et ਊl., 2009.). Heteromerizacija različitih podjedinica Kinovih kanala od velike je važnosti za povećanje funkcionalne raznolikosti i regulaciju različitih tipova stanica (Dreyer et ਊl., 1997. V éry i Sentenac, 2003. Xicluna et ਊl., 2007. Jeanguenin et ਊl., 2008 Lebaudy et ਊl., 2008 Lebaudy et ਊl., 2010). Iako je ovo ponašanje također predloženo za kanale vanjskog ispravljača (Kout), heteromerizacija je prijavljena samo među podjedinicama Kin ili Kout, sprječavajući stvaranje heteromernih struktura između dva tipa podjedinica (Dreyer et ਊl., 2004).

K + -Unosni nosači

Proteini obitelji KT/HAK/KUP prisutni su u biljkama, gljivama, bakterijama, pa čak i virusima (Greiner et ਊl., 2011 Santa-Maria et ਊl., 2018), a često su povezani s transportom K + preko membrane i opskrba K +. U bakterijskim genomima, nositelji K + ove obitelji kodirani su genima s jednom kopijom kup. U Escherichia coli, kup je konstitutivni sustav preuzimanja niskog afiniteta koji djeluje kao simpatizer K + -H + (Zakharyan i Trchounian, 2001). U gljivama su homologni proteini kodirani sa HAK1-slični geni prisutni u jednoj ili dvije kopije u većini vrsta. Za razliku od bakterijskog kup, gljivična HAK geni su snažno inducirani izgladnjivanjem K +, a kodirani proteini posreduju K + transport s visokim afinitetom (Benito et ਊl., 2011). U biljkama su ti transporteri poznati kao KT, HAK ili KUP (obitelj KT/HAK/KUP) i predstavljeni su s više gena u svojim genomima. Članovi ove obitelji bili su naširoko povezani s preuzimanjem K + s visokim afinitetom iz tla, dok drugi mogu djelovati i u transportu s niskim i/ili visokim afinitetom (Luan et ਊl., 2009. Very et ਊl., 2014. ) i druge uloge povezane, na primjer, s K + translokacijom, kontrolom kretanja vode na biljnoj razini, tolerancijom soli, osmotskim reakcijama/reakcijama suše, transportom drugih alkalnih kationa i razvojnim procesima u biljkama, kao što su rast korijena i auksin distribucija (Li et ਊl., 2018. Santa-Maria et ਊl., 2018.). Ove različite funkcije transportera KT/HAK/KUP mogu proizlaziti iz njihove kritične uloge u staničnoj K + homeostazi. KT/HAK/KUP geni nisu prisutni u životinjskim stanicama, što bi moglo ukazivati ​​da su oni ključni za transport K + u organizmima koji se suočavaju s vanjskim otopinama s fluktuirajućom i vrlo niskom koncentracijom K +, često u rasponu μM (Ashley et ਊl., 2006.). Na temelju dosadašnjeg znanja, KT/HAK/KUP geni su prisutni u svim biljnim genima, što je u suprotnosti s onima u drugim kraljevstvima, gdje su prisutni samo u određenim vrstama (Grabov, 2007 Greiner et ਊl., 2011). Ova razlika može odražavati važnost ovih transportera za biljni način života.

KT/HAK/KUP transporteri kopnenih biljaka razvrstani su prema homolognosti slijeda u šest grozdova ili klasa (I –VI), pri čemu klasa VI uključuje samo članove briofita (Santa-Maria et ਊl., 2018.). Filogenetska analiza pokazuje da se svi KT/HAK/KUP-ovi iz algi razlikuju od klasa kopnenih biljaka, što sugerira da se diverzifikacija u te skupine dogodila nakon kolonizacije zemljišta zelenim organizmima (Santa-Maria et ਊl., 2018.). Grupa KT/HAK/KUP iz kritosjemenjača prikazuje veliki i prilično promjenjiv broj članova u različitim genomima biljnih vrsta koji su do sada bili sekvencirani. Na primjer, postoji 13 gena Arabidopsis, 16  in breskve, 17  in vinove loze, 20  in Medicago, 21  in Manioka, 27   u riži, kukuruzu i Brachypodium, i 57  in Panicum virgatum (Song et ਊl., 2015. Nieves-Cordones et ਊl., 2016a, c Ou et ਊl., 2018). Članovi obitelji KT/HAK/KUP u kritosjemenici svrstani su u klase I –V (Nieves-Cordones et ਊl., 2016c).

Transporteri KT/HAK/KUP koji sudjeluju u preuzimanju K + iz tla grupirani su u zasebnu podgrupu klase I, nazvanu Ia (Nieves-Cordones et ਊl., 2016c) i koju ovdje nazivamo transporterima sličnima HAK1. kolege gljivica. Ova podgrupa uključuje ječam HvHAK1 (Santa-Mar ໚ et ਊl., 1997. Gierth i M äser, 2007.), Arabidopsis AtHAK5 (Rubio et ਊl., 2000 Gierth et ਊl., 2005), riža OsHAK1 i OsHAK5 (Ba ñuelos et ਊl., 2002), papar CaHAK1 (Martinez-Cordero et ਊl., 2004), rajčica LeHAK5 (Nieves-Cordones et ਊl., 2007.), i Thellungiella ThHAK5 (Alem án et ਊl., 2009b). K + transport s visokim afinitetom prikazan je za sve transportere slične HAK1 u heterolognim ekspresijskim sustavima (Nieves-Cordones et ਊl., 2014a). S druge strane, AtKUP7, koji pripada klasi V, mogao bi biti uključen u preuzimanje K + od niske do umjerene vanjske koncentracije K + (Han et ਊl., 2016.), a time i sudjelovanje u preuzimanju K + iz tla proteina iz različite klade ne treba odbaciti. Zanimljivo je da bi drugi članovi klase I   mogli biti povezani s preuzimanjem K + od strane stanica iz specijaliziranih tkiva. DmHAK5 iz venerinih muholovki uključen je u preuzimanje K + oslobođenog iz probavljenog plijena u dvokrilnom organu za hvatanje (Scherzer et ਊl., 2015.), dok kinoa slična CqHAK5 tjera K + dotok u stanice soli lista mjehura kako bi pridonijeli osmotskoj ravnoteži citosola u odnosu na osmotski tlak vakuola koje sadrže sol (Bohm et ਊl., 2018).

KT/HAK/KUP transporteri filogenetski su povezani s nadporodicom transportera kiselina-poliamin-organokacija (APC) koja sadrži sekundarne aktivne transportne proteine ​​odgovorne za uniport, simport i antiport širokog spektra supstrata (Vastermark et ਊl., 2014 ). Uzimajući kao predložak kristalne strukture prokariotskih APC transportera, računalno 3D modeliranje AtKUP7 (Ahn et ਊl., 2004. Al-Younis et ਊl., 2015. Santa-Maria et ਊl., 2018.), AtKUP4/TRH1 (Daras et &# x000A0al., 2015.), OsHAK1 (Rai et ਊl., 2017.), AtKUP1/TRH1 (Santa-Maria et ਊl., 2018.), AtHAK5 (Santa-Maria et ਊl., 2018.) i HvHAK1 (Santa- Maria et ਊl., 2018.). Strukturni modeli (slika 2) pokazuju prisutnost zajedničkih svojstava među svima njima: (1) hidrofobna jezgra koja sadrži 10 segmenata transmembrane (TM) i 10 (2) tri citosolne domene & N-i C-završetak i regija koja sadrži približno 70 ostataka smještenih između druge i treće TM (petlja II –III). Iako struktura pore još nije opisana, nekoliko je radova analiziralo učinak mutacija na funkciju ovih transportera (Santa-Maria et ਊl., 2018). Do sada su rezultati ukazivali da nekoliko dijelova proteina može pridonijeti postavljanju Vmax transportera i da regija koja uključuje od N-kraja do petlje II –III može pridonijeti određivanju njegovog Km. Nadalje, poravnanja sekvenci pokazuju da, iako nema opsežnog očuvanja sekvenci, 40 aminokiselinskih ostataka sačuvano je na potpuno istom položaju u svim eukariotskim transporterima HAK-a i u bakterijskim transporterima Kup (Rodriguez-Navarro, 2000). Šest od ovih očuvanih ostataka uključeno je u izrazito očuvan motiv u prvu transmembransku domenu čiji je konsenzusni slijed GVVYGDLGTSPROMETOVATI (aminokiseline sačuvane u svim proteinima su podebljane) (Rodriguez-Navarro, 2000). Prikaz ovog transmembranskog fragmenta sa spiralnim kotačem locira tri glicinska ostatka na istoj strani spirale, koja u slučaju tetramerne strukture može djelovati kao filter selektivnosti supstrata analogno GXGYGD motivu visoko očuvanom u K + kanalima. S tim u vezi, predloženo je da AtKUP4/TRH1 može tvoriti homodimere (Daras et ਊl., 2015.), što vjerojatno uključuje interakciju između domena C-kraja i manje vjerojatno između petlji II –III.

Za razliku od VG kanala koji su svi usmjereni na plazma membranu, zabilježeni su transporteri KT/HAK/KUP u različitim podstaničnim odjeljcima (Tablica 1). Većina karakteriziranih transportera obitelji KT/HAK/KUP nalazi se u plazma membrani, iako nisu svi uključeni u prehranu K +. Na primjer, čini se da AtKUP4/TRH1 sudjeluje u transportu auksina povezanom s gravitropizmom korijena i razvojem korijena dlake (Rigas et ਊl., 2013), dok AtKUP6 djeluje u pokretanju i razvoju korijena na signalnim putovima auksina i ABA unakrsnog razgovora (Osakabe et & #x000A0al., 2013.).

stol 1. Odabrano podstanično mjesto Arabidopsis, ječam, riža i Physcomitrella patens Transporteri KT/HAK/KUP.

HKT proteini

Bogat repertoar transportera kodiranih u genomu biljaka uključuje proteine ​​koji su zajedno poznati kao K + transporteri visokog afiniteta (HKT -ovi slika 2) unatoč činjenici da ti proteini omogućuju Na + -selektivni uniport ili Na + -K + simort sa kanalom -slična aktivnost (Benito et ਊl., 2014.). Filogenetske i funkcionalne analize razlikovale su dvije HKT podfamilije (Platten et ਊl., 2006). Članovi potporodice I   (HKT1) prisutni su u biljkama, Na + -selektivni, i uglavnom su uključeni u recirkulaciju Na + kroz vaskularna tkiva, što najbolje ilustrira AtHKT11 (Sunarpi et ਊl., 2005). Pripadnici potporodice II (HKT2) pronađeni su samo u monokotiledonim vrstama. Iako su svi propusni za K +, mehanički HKT2 mogu djelovati ili kao simpatizeri Na + -K + ili kao K + -selektivni uniporteri [pregledao Benito et ਊl. (2014)]. Proteini žitarica slični HKT2 uključeni su u prehranu K +.


Objašnjenje zašto više iona Ca2+ uzrokuje dulju duljinu korijena u biljkama - Biologija

Jedna od karakterističnih karakteristika svih živih organizama je ta da sadrže karakterističnu mješavinu iona i malih molekula. Sastav se ne samo razlikuje od okoliša, već se može razlikovati i unutar stanice. Na primjer, koncentracija vodikovih iona u nekim staničnim odjeljcima može biti 104 puta veća nego u drugima (mitohondriji koji dosežu pH čak 8 lizosoma s pH nižim od 4, BNID 107521, 106074). Omjer koncentracija iona Ca 2+ u odjeljcima izvanstanične i unutarstanične tekućine može ponovno biti 104 puta (BNID 104083). Ta je koncentracijska razlika toliko velika da transport iona Ca 2+ preko membrane, od unutar- do izvanstaničnog odjeljka, zahtijeva energiju više od jednog protonskog ili natrijevog iona koji teče niz gradijent protonske sile. Da bi se ovo uvjerilo, čitatelj bi se trebao sjetiti općeg pravila iz naših trikova sa trgovačkog popisa da je za uspostavljanje reda potencijala razlike potencijala potrebno 6 kJ/mol (≈2 kBT). Ta se energija može postići, na primjer, transportom jednog električnog naboja kroz razliku potencijala od 60 mV. Za postizanje četiri reda omjera koncentracije tada bi naboj trebao putovati oko 240 mV pokretne sile elektrona (zapravo čak i više zbog dvostrukog naboja kalcijevog iona). To je vrlo blizu naponu proboja membrane kako je objašnjeno u vinjeti na temu "Kolika je razlika električnog potencijala kroz membrane?". Doista, visoki omjer koncentracije Ca 2+ obično se postiže spajanjem na transport tri natrijeva iona ili hidrolizom ATP -a, što pomaže u postizanju potrebne razlike u gustoći bez opasnog aktiviranja membrane.

Drugi zakon termodinamike uči nas da će općenito prisutnost gradijenata koncentracije na kraju biti uklonjena procesima transporta mase, koji sustave stalno dovode u stanje ravnoteže. Međutim, iako nam drugi zakon termodinamike govori o prirodi krajnjeg stanja sustava (npr. Jednolike koncentracije), ne govori nam koliko će vremena trebati da se to stanje postigne. Membrane su evoluirale tako da tvore vrlo učinkovitu prepreku spontanom prijenosu mnogih ionskih i molekularnih vrsta. Da bismo procijenili vremensku skalu za izjednačavanje koncentracija, moramo znati brzine prijenosa mase, koje ovise o ključnim svojstvima materijala, poput konstanti difuzije i propusnosti.

Izuzetno uspješna klasa "zakona", koji opisuju ponašanje sustava koji su pretrpjeli mali odmak od ravnoteže, su linearni zakoni transporta. Ovi zakoni postavljaju jednostavan linearni odnos između brzine prijevoza određene količine interesa i pripadajuće pokretačke sile. Za transport mase postoji linearna veza između toka (tj. Broja molekula koje prelaze jedinicu površine po jedinici vremena) i razlike koncentracije (koja služi kao relevantna pokretačka snaga). Za transport preko membrana ove su ideje kodificirane u jednostavnoj jednadžbi (za neutralnu otopljenu tvar) j = – p · (cu-cvan), gdje je j neto protok u ćeliju, cu i cvan odnose se na koncentracije unutar i izvan područja vezanog za membranu, a p je parametar materijala poznat kao propusnost. Jedinice p mogu se zaključiti ako se primijeti da tok ima jedinice broja/(površina x vrijeme) i da koncentracija ima jedinice broja/volumena, što znači da su same jedinice p duljina/vrijeme. Kao i mnoge transportne količine (npr. Električne vodljivosti materijala koje prelaze 30 redova veličine), propusnost ima vrlo veliki dinamički raspon kako je prikazano na slici 1. Kao što se vidi na slici, lipidni dvoslojevi imaju gotovo 10 10 -kratni raspon propusnosti.

Slika 1: Širok raspon membranskih propusnosti različitih spojeva u stanici. Membrane su propusnije za nenabijene spojeve i najmanje propusne za nabijene ione. Imajte na umu da će postojanje ionskih kanala učiniti očiglednu propusnost kada su otvoreni nekoliko redova veličine. Jedinice su odabrane kao nm/s, a nekoliko nm je karakteristična širina membrane. Slika prilagođena prema RN Robertson, The Lively Membranes, Cambridge University Press, 1983. Vrijednost glukoze je manja nego u Robertsonu na temelju nekoliko izvora kao što su BNID 110830, 110807. Ostali izvori podataka: BNID 110729, 110731, 110816, 110824, 110806.

Koji fizikalno-kemijski parametri vode do smještanja spoja na ovoj ljestvici propusnosti? Zlatno pravilo je da male molekule imaju veću propusnost od većih molekula. Još jedno opće pravilo je da neutralni spojevi mogu prijeći membranu mnogo redova brže od sličnih nabijenih spojeva. Među nabijenim spojevima, negativni (anionski) spojevi imaju tendenciju imati mnogo veću propusnost od pozitivnih (kationskih) spojeva. Takozvano Overtonovo pravilo kaže da se propusnost membrane povećava s hidrofobnošću, gdje je hidrofobnost sklonost spoja da preferira nepolarno otapalo nad polarnim (vodenim) otapalom. Overtonovo pravilo predviđa da će nabijene molekule (nehidrofobne), poput iona, imati nisku propusnost jer podnose energetsku kaznu povezanu s prodorom kroz membranu, dok otopljeni plinovi poput O2 i CO2, koji su hidrofobni (budući da su nenabijeni i simetrični), imat će visoku propusnost. Doista, propusnost dvoslojnih membrana lipida za CO2 daju vrijednosti koje su 0,01-1 cm/s (da, mjerenja propusnosti imaju vrlo velike nesigurnosti među različitim laboratorijima, BNID 110004, 110617, 102624), veće od svih ostalih vrijednosti prikazanih na slici 1. Ova vrijednost pokazuje da barijera koju stvara stanična membrana zapravo je manja prepreka od barijere uzrokovane nemirnim slojem vode koji zahvaća staničnu membranu izvana. Takav zaključak može se izvesti jednadžbom za koeficijent propusnosti prepreke, danom s p = K x D/l gdje je l širina, D koeficijent difuzije i K koeficijent podjele između medija i materijala prepreke. Ovo je također poznato kao model "topljivosti-difuzija" za propusnost gdje oni označavaju efekte K i D koji su dva koraka koji utječu na propusnost. Za nemirni sloj vode K = 1 jer je vrlo sličan mediju, ali za membranu vrijednost za sve osim za najviše hidrofobnog materijala obično je nekoliko redova veličine manja od 1. Ova ovisnost o K je u središtu Overtonove gore spomenuto pravilo. Visoka propusnost za CO2 također sugerira da kanali poput akvaporina za koje se predlaže da služe za transport plina u stanicu nisu potrebni jer je membrana dovoljno propusna. Da bismo vidjeli kako svojstva membrane utječu na kemijski sastav metabolita, okrećemo se izračunavanju vremena curenja za različite spojeve

Slika 2: Zadnji dio izračuna omotača vremenskog okvira za nefosforiliranu molekulu glukoze koja će pasivno difundirati iz bakterijske stanice. Funkcionalne implikacije se zatim razmatraju za ćelije koje brzo rastu gdje je učinak zanemariv i za stanice u stacionarnom stanju gdje mogu uzrokovati značajno curenje resursa.

U obzir uzimamo glicerol, na primjer. Analiza prikazana na slici 2 daje procjenu vremena njezina istjecanja iz stanice ako molekula nije fosforilirana ili na drugi način pretvorena u hidrofilniji oblik. Propusnost stanične membrane za glicerol je p≈10-100 nm/s (BNID 110824) kako se može iščitati sa slike 1. Vremenska skala da molekula glicerola unutar stanice pobjegne natrag u okolni medij, pod pretpostavkom da nema povratka ulije u ćeliju (cvan= 0), može se grubo procijeniti primjetom da je istjecanje iz ćelije p · A · cu gdje je A površina stanice. Vremenska skala se nalazi uzimajući ukupnu količinu u ćeliji, V · cu (where V is cell volume or more accurately the cell water volume), and dividing by this flux resulting for a bacterial cell (r≈1 μm) in a time scale:

This is a crude estimate because we did not account for the decreasing concentration of cu with time that will give a correction factor of 1/ln(2), i.e less than 2 fold increase. What we learn from these estimates is that if the glycolytic intermediates glyceraldehyde or dihydroxyacetone which are very similar to glycerol were not phosphorylated, resulting in the addition of a charge, they would be lost to the medium by diffusion through the cell membrane. In lab media, where a carbon source is supplied in abundance, this is not a major issue, but in a natural environment where cells are often waiting in stationary phase for a lucky pulse of nutrients (E. coli is believed to go through months of no growth after its excretion from the body before it finds a new host), the cell can curb its losses by making sure metabolic intermediates are tagged with a charge that will keep them from recrossing the barrier presented by the lipid bilayer.


Explanation of why more Ca2+ ions causes longer root length in plants - Biology

A previous blog entry, Healthy Soils for Healthy Trees, discussed the importance of preserving soil structure from being destroyed by compaction. Together, soil texture and soil structure have the greatest influence on pore space in soil, and how easily air, water, and roots can move through a soil. Many people are aware of what soil texture – proportions of sand, silt and clay – they are dealing with on a site. Few people consider a soil’s structure, though, even though in most soils, the structure is just as important as the texture. Two soils with the same texture can behave very differently depending on their structure. A clay soil, for example, can be easy for air, water, and roots to move through with good structure, or be almost impenetrable by roots, air, and water when its structure has been destroyed by compaction.

How soil structure develops

Soil structure refers to how particles of soil are grouped together into aggregates (also called peds). They are cemented or bound together by physical, chemical, and biological processes.

Physical-chemical processes that build soil structure include:

  • Polyvalent cations like Ca2+, magnesium Mg2+, and aluminum Al3+ bind together clay particles
  • Soil particles are pushed closer together by freezing and thawing, wetting and drying, and by roots pushing through the soil as they grow in length and width.

Biological processes that build soil structure include:

  • Soil particles are cemented together by humus, by organic glues created by fungi and bacteria decomposing organic matter, and by polymers and sugars excreted from roots.
  • Fungal hyphae and fine roots stabilize aggregates (University of Minnesota Extension 2002.)

Organic matter and plant roots are therefore key to soil structure.

How soil structure deteriorates

Factors that can deteriorate or destroy soil structure include, for example:

  • Compaction
  • Cultivation
  • Removal of vegetation
  • Excessive moving and handling of soil
  • Screening
  • Excessive sodium

A high proportion of sodium to calcium and magnesium causes clay particles to repel each other when wet, so aggregates are dispersed and the process of soil structure formation is reversed. Soils with too much sodium become almost impermeable to water because the dispersed clay and small organic particles clog up remaining soil pores (Donahue et al 1983). Excessively high sodium levels can result from irrigation and salting roads.

Different types of soil structure

Soil structure is classified by type (shape), class (size) of peds, and grade (strength of cohesion) of aggregates. The shape, size, and strength of aggregates determine pore structure, and how easily air, water, and roots move through soil (Donahue et al 1983).

Figure 1 shows the different types of soil aggregates, and how easily water typically moves through each of these types.

Figure 1: Types of soil aggregates (Image from Victorian Resources)

Granular structure is the most common in surface soil layers, especially those with adequate organic matter. Granular structures offer the most pore space of any structure (Cooperative Soil Survey, no publication date given).

Image from Victorian Resources

Columnar structure is often found in soils with excessive sodium, due to the dispersing effects of sodium, which destroys soil structure, rendering the soil effectively sealed to air and water movement (Cooperative Soil Survey, no publication date given).

Image from Victorian Resources

Platy structure has the least amount of pore space and is common in compacted soils (Cooperative Soil Survey, no publication date given).

Image from Victorian Resources

Some soils have no true structure, like single grain soils (like a loose sand with little to no attraction between the grains of sand), and massive soils (large cohesive masses of clay).

Image from Victorian Resources

For more information on soil structural classification, see the resources listed in the references section below.

Ways to preserve desirable soil structure

As the USDA Natural Resources Conservation Service (2008) explains: “practices that provide soil cover, protect or result in the accumulation of organic matter, maintain healthy plants, and avoid compaction improve soil structure and increase macropores.”

Other key practices to preserve soil structure include eliminating soil screening and minimizing handling, and avoiding the use of sodium salts.

Implications for bioretention

Preserving soil structure may increase the range of soil textures acceptable for bioretention. Bioretention soils are often sand-based, primarily to ensure adequate infiltration rates. Clay and silt content is often limited to a maximum of only 3 to 5 percent, which is very, very low, limiting soils to sands according to the soil textural triangle. While a clay soil that has been screened and has no structure will have a very low infiltration rate, with proper structure, many soils with more clay can also have adequate infiltration rates. Increasing clay content above the very low maximum of 3 to 5 percent could provide important benefits, including increased soil water holding capacity and increased cation exchange capacity, which increase potential pollutant removal. When increasing clay content, however, keep in mind that the higher the clay content, the more crucial it becomes to protect soil from compaction and from excess salt, as clay soils are more prone to compaction and loss of structure, and unacceptable decrease of infiltration rates due to dispersion from sodium ions.


Rezultati

Expression of CDPK Genes

There are 34 CDPK genes in the Arabidopsis genome [35,46]. To investigate whether and, if so, where CDPKs may function in ion channel regulation and guard cell signal transduction branches, we first identified CDPK genes expressed in guard cells using a guard cell–enriched cDNA library and RT-PCR with degenerate oligomers [55]. Two of the guard cell–expressed CDPK genes, CPK3 (AGI No.: At4g23650) and CPK6 (AGI No.: At2g17290), showed initial insertion mutant phenotypes and were therefore further analyzed. The expression of CPK3 i CPK6 in isolated guard cell protoplasts (GCPs) was further analyzed by RT-PCR with gene-specific primers (Figure 1A) and independently later by cell type–specific genomic scale expression analyses using Affymetrix (Santa Clara, California, United States) GeneChip assays [54]. RT-PCR analysis showed that CPK3 i CPK6 are expressed in both guard cells and mesophyll cells (Figure 1A). The purity of GCPs was analyzed by RT-PCR with specific primers for the guard cell–expressed potassium channel gene, KAT1 (Figure 1A) [56]. Guard cell preparations were further examined for contamination of mesophyll cells by analyzing mRNA abundance of a putative calmodulin-binding protein (CBP) (AGI No.: At4g33050), which was identified as being highly expressed in mesophyll cells but absent in guard cells [54]. No CBP mRNA was detected in guard cell preparations (Figure 1A), indicating that the GCP preparations had no or very little contamination. In addition to CPK3 i CPK6, several other CDPK genes were identified in guard cells by microarray experiments with guard cell RNA (Supplemental Table I in [54]). In this study, we focus on functional dissection of the guard cell–expressed CPK3 i CPK6 genes.

(A) Expression of CPK3 i CPK6 in guard cell (GC) and mesophyll cell (MC) protoplasts was examined by RT-PCR. Control amplifications of the guard cell-expressed KAT1 gene and the mesophyll-expressed CBP marker genes [54] (Leonhardt et al., 2004) were used to test the purity of cell preparations (see Results). ACTIN2 was used for an internal loading control. To amplify each CDPK-specific band, RT-PCR was performed with primer sets as indicated by arrowheads in (B) for 36 cycles. Plants were sprayed with water (−ABA) or 100 μM ABA (+ABA) 4 h before isolation of protoplasts and RNA extraction.

(B) Cartoon showing the T-DNA insertion positions in cpk3 i cpk6 T-DNA insertion alleles. PCR was performed with a left boarder primer of the T-DNA and a gene-specific primer, and the PCR products were sequenced to determine the T-DNA insertion positions. Arrowheads indicate primer locations for RT-PCR in (A) and (C). ATG and TGA indicate start and stop codons. White boxes indicate exons.

(C) RT-PCR confirmed that cpk3–1 i cpk6–1 alleles were disruption mutants. PCRs (32 cycles) were performed with primer sets as indicated in (B) (black arrowheads) in the left three panels. Transcripts of wild-type (WT) and cpk6–2 were examined with two sets of primers [white and black arrowheads in (B)] showing that cpk6–2 lacks exon 1 and that the cpk6–2 has 8% or less the mRNA level of wild-type based on densitometry analyses (n = 2). RNA was extracted from leaves of WT, homozygous cpk3–1, cpk6–1, i cpk6–2 single mutants, and the cpk3-1cpk6–1 double mutant.

To genetically analyze functions of CPK3 and CPK6 in guard cell signal transduction, we identified T-DNA insertion mutations in CPK3 i CPK6 from the Salk Institute Genomic Analysis Laboratory database [57]. Homozygous T-DNA insertion mutant lines were isolated and genomic sequences of the cpk3–1 (SALK_107620), cpk3–2 (SALK_022862), cpk6–1 (SALK_093308), and cpk6–2 (SALK_033392) insertion mutants were determined. The T-DNA insertions in cpk3–1 i cpk3–2 are localized in the first exon and in the first intron, respectively (Figure 1B). The insertion in cpk6–1 is localized in the second exon, 60 base-pairs downstream of the translation initiation codon, and cpk6–2 is in the first intron (Figure 1B). Southern blot analyses of homozygous plants indicated only a single band in each line, suggesting a single T-DNA insertion in these mutants (data not shown). Transcripts of CPK3 ili CPK6 were not detected in cpk3–1 i cpk6–1 as demonstrated by RT-PCR utilizing whole leaf RNA extracts (Figure 1C). No RT-PCR band was observed for cpk3–2 (data not shown). Za cpk6–2, no full-length cDNA was detected (Figure 1C). A faint band for transcript downstream of the T-DNA insertion was observed after 35 cycles of amplification showing substantially reduced mRNA levels (8% or less intensity compared to wild-type level, n = 2) (Figure 1C). Using a primer set in the first exon and the eighth exon, no RT-PCR amplification was observed (Figure 1B and 1C), showing that the first exon is missing in cpk6–2. We performed RT-PCR with CPK3 primers in cpk6–1 and with CPK6 primers in cpk3–1 to examine whether a compensatory expression occurs. No compensation in the wild-type transcript levels was observed (data not shown).

Homozygous cpk3 i cpk6 single and cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutants were isolated and used for further analyses. Whole plant general morphological phenotypes of cpk3–1, cpk3–2, cpk6–1, cpk6–2, i cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutants were largely similar to wild-type plants (Columbia ecotype) under the standard growth conditions tested, but cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutant plants showed a slight delay in growth by approximately 2 d in 4-wk-old plants compared to wild-type plants (data not shown).

Activation of S-Type Anion Channels by Cytosolic Ca 2+ Is Impaired in cpk3cpk6 Mutants

S-type anion efflux channels have been proposed to play an important role as targets of ABA signal transduction in guard cells and to be regulated by upstream phosphorylation events [5,9,16,32,55,58–62]. To determine whether CDPKs function in the activation of S-type anion channels in guard cells, we examined Ca 2+ activation of S-type anion channels in wild-type, cpk3, i cpk6 single mutant and double mutant guard cells.

As illustrated in Figure 2, typical large [Ca 2+ ]cyt-activated S-type anion channel currents were observed in wild-type guard cells in the presence of elevated (2 μM) free Ca 2+ in the patch pipette (cytosolic) solution (Figure 2A and 2C) as previously described [5,9]. At 0.1 μM [Ca 2+ ]cyt in the pipette, large S-type anion currents were not activated (data not shown) [9]. S-type anion channel currents in the presence of 2 μM [Ca 2+ ]cyt were significantly reduced in the cpk3–1 mutant (n = 11 guard cells) compared to wild-type (Figure 2C n = 17, str = 0.022 at −145 mV). cpk3–2 showed similar results to cpk3–1 (Figure 2C n = 7, str > 0.4 compared to cpk3–1). S-type anion channel currents were further reduced in cpk6–1 (n = 11) and cpk6–2 (n = 4) single mutant guard cells (Figure 2C str < 0.005 compared to wild-type str > 0.18 for cpk6–1 compared to cpk6–2). U cpk3-1cpk6–1 double mutant guard cells (n = 17), S-type anion channel currents were substantially reduced compared to wild-type controls (Figure 2B and 2C str < 0.0005). Essentially similar results were obtained in cpk3-2cpk6–2 (Figure 2C n = 6, str > 0.4 compared to cpk3-1cpk6–1). Nevertheless, background anion currents did remain in the double mutants (Figure 2B and 2C). The data showed stronger effects of CPK6 disruptions compared to CPK3 disruptions. Thus CPK3 i CPK6 are important for Ca 2+ activation of S-type anion channels.

(A and B) Typical S-type anion channel current traces in wild-type (WT) (A) and cpk3-1cpk6–1 double mutant (B) guard cells are shown in response to 2 μM free Ca 2+ in the patch-clamp pipette solution that dialyzes the cytoplasm.

(C) Average current-voltage curves of wild-type (n = 17 cells), cpk3–1 (n = 11), cpk3–2 (n = 7), cpk6–1 (n = 11), cpk6–2 (n = 4), cpk3-1cpk6–1 (n = 17), and cpk3-2cpk6–2 (n = 11). Error bars show SEM.

Impairment in ABA Activation of S-Type Anion Channels in cpk3cpk6 Mutants

Previous studies have shown that guard cell signal transduction is mediated by a network of events, which includes parallel Ca 2+ -dependent and -independent signaling branches (see Discussion for reviews: [3,4]). Therefore, experiments were pursued to analyze ABA activation of S-type anion channels. These experiments were performed under different conditions than those shown in Figure 2, such that cytosolic Ca 2+ elevation alone would not fully activate S-type anion currents (see Materials and Methods) [9]. As shown in Figure 3, with preincubation of guard cells in low extracellular Ca 2+ , 2 μM cytosolic Ca 2+ in the pipette activated S-type anion currents in guard cells of only intermediate amplitudes (Figure 3A). Under these conditions, ABA up-regulated S-type anion current activities in wild-type protoplasts (Figure 3A, 3B, and 3E n = 8). U cpk3-1cpk6–1 (n = 7) and cpk3-2cpk6–2 (n = 7) double mutant guard cells, ABA regulation of S-type anion currents was impaired (Figure 3C, 3D, and 3E str < 0.01). These data show that, despite the complex network of ion channel regulation mechanisms in guard cells [3], CDPKs mediate an important Ca 2+ -decoding transduction step in ABA regulation of S-type anion channels (Figures 2 and 3). ABA activation in wild-type guard cells and the impairment in ABA activation of S-type anion channels at 2 μM cytosolic Ca 2+ in cpk3cpk6 mutants (Figure 3) further provide evidence for a recently proposed hypothesis in which stomatal closing signals (i.e., ABA) mediate priming of guard cell Ca 2+ sensors, such that they can respond to elevated cytosolic Ca 2+ levels [63].

(A and B) Whole-cell S-type anion channel current traces in wild-type (WT) guard cells in the absence of ABA (A) and in the presence of 50 μM ABA (B).

(C and D) Whole-cell S-type anion channel current traces in cpk3-1cpk6–1 double mutant guard cells in the absence of ABA (C) and in the presence of ABA (D).

(E) Average current-voltage curves of wild-type and cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutant guard cells in the absence and presence of ABA (n = 8 WT n = 7 cpk3-1cpk6–1 n = 3 cpk3-2cpk6–2 guard cells). GCPs were treated with 50 μM ABA or solvent control (0.1% ethanol) for 2 h prior to establishing gΩ seals. Open and closed symbols indicate −ABA and +ABA, respectively.

Impairment in ABA activation of ICa Channels in cpk3 cpk6 Mutants

ABA activates plasma membrane Ca 2+ -permeable (ICa) channels [64–66]. Combined physiological, molecular genetic, and cell biological analyses have shown that ICa channels function in the guard cell ABA signal transduction network at hyperpolarized voltages [9,53,64–66]. We examined whether CPK3 i CPK6 function in the regulation of ICa channels.

Typical ICa currents were activated by extracellular application of ABA to patch-clamped wild-type guard cells (Figure 4A and 4B, n = 13). Unexpectedly, ABA activation of ICa channels was not observed in cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutant guard cells (Figure 4C–4E n = 14, str = 0.56 for cpk3-1cpk6–1 n = 8, str = 0.47 for cpk3-2cpk6–2 when comparing before and after ABA treatment). Blind patch-clamp experiments in which the genotype of protoplasts was unknown (n = 2 for wild-type, n = 2 for cpk3-1cpk6–1), and similar findings by Y.M., I.C.M., Y.W., and S.M. in this study, further confirmed the impairment of ABA activation of ICa channels in cpk3cpk6 mutant guard cells. Next we analyzed whether only one of the two CDPKs might affect ABA activation of ICa channels. Defects in the ABA activation of ICa channels were observed in the cpk3–1, cpk3–2, cpk6–1, i cpk6–2 single mutants (Figure 4F–4I n = 9, str = 0.38 for cpk3–1 n = 11, str = 0.47 for cpk6–1 n = 3, str = 0.96 for cpk3–2 n = 5, str = 0.30 for cpk6–2, when comparing before and after ABA-treatment). Together these data show that CPK3 i CPK6 function in ABA regulation of ICa channels (Figure 4) and Ca 2+ and ABA activation of S-type anion channels (Figures 2 and 3).

(A and B) ABA (50 μM) activated ICa channel current in wild-type (WT) guard cells. (A) Current traces before (−ABA) and after ABA (+ABA) activation of ICa channels are shown in a representative cell. (B) Average current-voltage curves (n = 13) are shown.

(C and D) ABA failed to activate ICa channel currents in cpk3-1cpk6–1 double mutant guard cells. (C) A response in a representative cell is shown. (D) Average current-voltage curves (n = 14) are shown. Traces before and after ABA overlap in (C).

(E–I) Averages of current-voltage curves in guard cells isolated from (E) cpk3-2cpk6–2 double mutant (n = 8), (F) cpk3–1 (n = 11), (G) cpk6–1 (n = 9), (H) cpk3–2 (n = 3), and (I) cpk6–2 (n = 5) are shown.

(J and K) ABA activation of ICa channel currents in wild-type (WT) guard cells pretreated with the protein kinase inhibitor K252a (2 μM) for 15 min prior to and during patch clamping and with no ATP added in the patch pipette solution. Response in a guard cell is shown in (J), and average current-voltage curves (n = 6) are shown in (K).

Open symbols indicate −ABA and closed symbols indicate +ABA. Error bars represent SEM.

To gain insight into the question whether activation of phosphorylation events are required before or after ABA application and during ABA activation of ICa channels in patch-clamped Arabidopsis guard cells, wild-type guard cells were pretreated with the broad serine/threonine kinase inhibitor, K252a [(8R*,9S*,11S*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11,epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cyclo-octa[c,d,e]-trinden-1-one], 20 min prior to, and continuously during, patch clamping. Moreover, the cytoplasm of whole cells was dialyzed in the absence of ATP in whole cell recordings for longer than 15 min prior to extracellular ABA exposure. Ionic currents were recorded in the same guard cells prior to and after exposure to ABA. In negative controls, treatment with K252a alone did not cause constitutive activation of ICa channel currents without ABA treatment (n = 3). Interestingly, pretreatment with K252a (2 μM) did not disrupt ABA activation of ICa channels (Figure 4J and 4K n = 6, str < 0.04 at −180 mV). These findings indicate that the effect of the cpk3 i cpk6 mutations on ABA activation of ICa channel currents are most likely not caused by direct ABA-induced upstream CDPK activation in the NADPH-dependent activation branch of ICa channels [66], but possibly by prior CDPK action (see Discussion).

We further examined whether the defect in ABA activation of ICa channels is due to impairment in reactive oxygen species (ROS) activation of ICa channels, as found for the ABA-insensitive mutants, gca2 i abi2–1 [65,66]. As shown in Figure 5, hydrogen peroxide activation of ICa channels was observed in cpk3cpk6 double mutant and wild-type guard cells (Figure 5 n = 7, str < 0.001 for cpk3-1cpk6–1 n = 4, str < 0.05 for cpk3-2cpk6–2 i n = 7, str < 0.01 for wild-type when comparing before and after H2O.2 treatment).

(A and B) Exogenous H2O.2 (5 mM) activated ICa channels in (A) wild-type (WT) (n = 9) and (B) cpk3-1cpk6–1 double mutant guard cells (n = 12). Error bars represent SEM.

ABA- and Ca 2+ -Induced Stomatal Closure

The findings that cytosolic Ca 2+ and ABA activation of S-type anion channels and ABA activation of ICa channels are impaired in cpk3cpk6 mutants led us to examine ABA-induced stomatal closure in these cdpk mutants. Application of 1 and 10 μM ABA induced a decrease in stomatal aperture in wild-type (Figure 6A, n = 13 experiments, 260 stomata). U kontrastu, cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutant stomata showed reduced ABA responses (Figure 6A, Table 1, n = 7 experiments, 140 stomata at 10 μM ABA: str < 0.01 for cpk3-1cpk6–1 versus wild-type n = 4 experiments, 120 stomata, str < 0.011 for cpk3-2cpk6–2). Furthermore, ABA-induced stomatal closing was partially impaired in all of the cpk3 i cpk6 single mutant alleles (str < 0.05) (Table 1). These results show roles for CPK3 i CPK6 in ABA-induced stomatal closure.

(A) ABA-induced stomatal closing (white bars: wild-type shaded bars: cpk3-1cpk6–1 double mutant black bars: cpk3-2cpk6–2 double mutant). Average data from representative experiments are shown (wild-type: WT, n = 13 experiments, 260 total stomata cpk3-1cpk6–1 double mutant: n = 7 experiments, 140 stomata cpk3-2cpk6–2 n = 4 experiments, n = 120 stomata).

(B) External Ca 2+ -induced stomatal closing (white bars: wild-type, shaded bars: cpk3-1cpk6–1 double mutant, black bars: cpk3-2cpk6–2 double mutant). Average data from representative experiments are shown (wild-type: WT, n = 9 experiments including 4 blind experiments, 180 total stomata cpk3-1cpk6–1 double mutant, n = 9 experiments including 4 blind experiments, 180 total stomata cpk3-2cpk6–2, n = 4 experiments, n = 80 stomata). Stomatal aperture widths are illustrated. Error bars represent SEM.

ABA-Induced Stomatal Closure in cpk3 i cpk6 Mutants

Extracellular Ca 2+ causes stomatal closing, by initiating repetitive cytoplasmic Ca 2+ elevations in guard cells [67–69]. Application of 100 μM CaCl2 to intact leaves closed wild-type stomata (Figure 6B). However, in the cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutants, stomatal closure was significantly attenuated compared to the wild-type response (Figure 6B, str < 0.01 for cpk3-1cpk6–1 i str < 0.05 for cpk3-2cpk6–2 at 100 μM external Ca 2+ ). External Ca 2+ -induced stomatal closing was also impaired in all four cpk3 i cpk6 single mutants (n = 60 to 100 stomata str < 0.05, data not shown).

To further evaluate Ca 2+ regulation of stomatal closure, we examined the effect of experimentally imposed [Ca 2+ ]cyt oscillations on stomatal closure in cpk3cpk6 double mutant plants [29–31,68]. A Ca 2+ oscillation pattern was applied to guard cells with a similar time pattern to those that cause long-term programmed stomatal closure (i.e., inhibition of stomatal reopening) in Arabidopsis [29]. Hypothesizing a contribution of additional CDPKs or other Ca 2+ transducers in these experiments, we applied a hyperpolarizing buffer with lower extracellular Ca 2+ concentrations (1 mM) and higher KCl concentrations (1 mM) than those used in previous research [29] with the goal of experimentally imposing weaker cytosolic Ca 2+ transients in guard cells (see Materials and Methods). Ca 2+ imaging experiments of guard cells showed that this protocol causes cytosolic Ca 2+ oscillations in cpk3-1cpk6–1 double mutant and in wild-type guard cells (Figure 7, inset). The average amplitudes of imposed [Ca 2+ ]cyt transients were similar in wild-type and cpk3-1cpk6–1 double mutant guard cells (str = 0.25 wild-type: 0.725 ratio units [RU] ± 0.043 SEM, n = 16 wild-type cells cpk3-1cpk6–1: 0.766 RU ± 0.04 SEM, n = 24 cpk3-1cpk6–1 guard cells). The average integrated total [Ca 2+ ]cyt ratio increase per period was also determined and found to be similar for wild-type and cpk3-1cpk6–2 guard cells (str = 0.46 wild-type: 8.535 RU * 0.1 min ± 0.497, n = 16 wild-type cells cpk3-1cpk6–1: 8.456 RU * 0.1 min ± 0.517, n = 24 cpk3-1cpk6–1 cells). These data are consistent with findings that external Ca 2+ -induced [Ca 2+ ]cyt elevations in guard cells include intracellular Ca 2+ release from guard cell organelles [67,69].

Four [Ca 2+ ]cyt transients (inset) were imposed in wild-type (WT) (n = 6 experiments) and cpk3-1cpk6–1 double mutant (n = 6 experiments) stomata. Individually mapped stomatal apertures were measured for the last 30 min before imposed [Ca 2+ ]cyt transients (before Time = 0) and for the ensuing 180 min at the indicated time points. Error bars represent SEM. Inset (top) shows imposed [Ca 2+ ]cyt transients in wild-type (blue trace) and in cpk3-1cpk6–1 (red trace) guard cells expressing Yellow Cameleon 3.6 (R.U.: ratio units).

Four Ca 2+ transients with a 10-min period, which induce long-lasting “Ca-programmed” stomatal closure [29], were applied to wild-type and cpk3cpk6 double mutant stomata. Wild-type stomata started closing immediately after the first Ca 2+ elevation was imposed and continued to show progressive Ca 2+ -reactive closing for longer than 40 min (Figure 7 n = 6 experiments, 47% closure). In contrast, Ca 2+ transient-induced closure of cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutant stomata was reduced (14% and 22 % closure, respectively) (Figure 7 n = 6 experiments, str < 0.01 for cpk3-1cpk6–1 versus wild-type and n = 3 experiments, str < 0.05 for cpk3-2cpk6–2 versus wild-type, str > 0.60 cpk3-1cpk6–1 protiv cpk3-2cpk6–2). Stomata of both wild-type and cpk3cpk6 double mutant guard cells remained closed during the ensuing 2 h and 20 min measurements, even though cells were extracellularly bathed in a typical “stomatal opening” solution containing 50 mM KCl and 0 mM CaCl2 and exposed to white light (Figure 7, from 40 to 180 min). Interestingly, the partial stomatal closure of cpk3cpk6 double mutants was also maintained during the ensuing 2 h and 20 min after Ca 2+ transients were terminated, i.e. a significant stomatal closure was observed at 180 min when compared with 0 min in both cpk3-1cpk6–1 i cpk3-2cpk6–2 (str < 0.01 for both mutants). Thus, the rapid Ca 2+ -reactive stomatal closure response is clearly impaired in the cpk3cpk6 double mutants, whereas the long-term Ca 2+ -programmed stomatal closure response [29–31] appears to be functional (Figure 7).

Seed germination analyses were also pursued with wild-type and the cpk3-1cpk6–1 i cpk3-2cpk6–2 double mutants. No significant difference in ABA inhibition of seed germination in wild-type and the cpk3cpk6 double mutant alleles was observed in the presence of 0, 0.3, 1, and 5 μM ABA after 3, 5, 7, and 11 d (unpublished data).

Rapid-Type Anion Channel Activity Is Not Greatly Altered in cpk3cpk6 Double Mutant

ABA also regulates a second class or mode of anion channels in guard cells, the rapid-type (R-type) anion channels [62,70–73]. Therefore, we also compared R-type anion channel current properties in wild-type and cpk3cpk6 guard cells (Figure 8). The selectivity for anions over cations of these ion currents (Figure 8A) in Arabidopsis guard cells was further analyzed. Replacing Ba 2+ with the impermeable cation tetraethylamonium in the bath solution did not affect these R-type inward currents, as previously shown [65]. Moreover, use of the impermeable anion gluconate in the pipette solution abolished the current (n = 3, data not shown), confirming that the recorded currents are R-type anion currents. Interestingly, in contrast to S-type anion channels (Figures 2 and 3), no significant difference was observed in the rapid anion channel activity between wild-type and cpk3-1cpk6–1 double mutant guard cells (Figure 8A and 8B wild-type, n = 7 cpk3-1cpk6–1, n = 7 str = 0.21 at peak current). Tako, cpk3cpk6 mutant guard cells did not significantly impair R-type anion currents, which correlated with the partial ABA-induced stomatal closing in cpk3cpk6 mutant guard cells (Figure 6, Table 1).

(A) Representative traces of R-type anion channel current in wild-type (black trace) and cpk3-1cpk6–1 double mutant (gray trace).

(B) Averages of peak currents of R-type anion channel currents in wild-type (white bar, n = 7) and cpk3-1cpk6–1 double mutant (black bar, n = 7 ± SEM) guard cells.


Explanation of why more Ca2+ ions causes longer root length in plants - Biology

2. End-of-chapter questions

1 If sucrose is actively loaded into a sieve tube, which combination of changes take place in the sieve tube?


2 Which of the following rows correctly describes the hydrostatic pressure of the two types of elements?

A Higherlight intensities are associated with higher temperatures.
B Thepalisade mesophyll cells have fewer air spaces than the spongy mesophyll cells.
C Theupper epidermis has fewer stomata.
D Theupper epidermis is more exposed to light.


4. Explain how water moves from:
a the soil into a root hair cell.
b one root cortex cell to another.
c a xylem vessel into a leaf mesophyll cell.


5. Name three cell types found in:
i xylem
ii phloem.
b State the functions of the cell types you have named.


b. Explain the relevance of these dimensions and ratios to transport in large multicellular organisms.

7. Arrange the following in order of water potential. Use the symbol > to mean 'greater than'.
dry atmospheric air mesophyll cell root hair cell soil solution xylem vessel contents

8. Figure a shows changes in the relative humidity of the atmosphere during the daylight hours of one day.


Lik b shows changes in the tension in the xylem of a tree during the same period.

11. The figure is a graph showing the relationship between rate of transpiration and rate of water uptake for a particular prlant.

a Define the term transpiration. [2]
b State the two environmental factors which are most likely to be responsible for the changes in transpiration rateshown in the figure. [2]
c Describe the relationship between rate of transpiration and rate of water uptake shown in the figure. [2]
d Explainthe relationship. [4]

12 The figure is a light micrograph of a transverse section through the leaf of marram grass (Ammophila), a xerophytic plant.

a Assuming the magnification of the micrograph is x 100, calculate the length of the sieve element. Show your working. [3]

15. Translocation of organic solutes takes place between sources and sinks.

a Briefly explain under what circumstances:

b as size increases, volume increases faster than surface area
therefore as size increases, the surface area : volume ratio decreases
can no longer rely on diff usion to satisfy transport needs

7 soil solution > root hair cell > xylem vessel contents
> mesophyll cell > dry atmospheric air

8 a the lower the relative humidity, the higher the tension/the lower the hydrostatic pressure, in the xylem
more evaporation from leaf (mesophyll cells) when low relative humidity
results in lower water potential in leaf (mesophyll cells)
therefore more water moves from xylem (vessels to replace water lost from leaf)
down a water potential gradient
sets up tension in the xylem vessels

b lowest/most negative, hydrostatic pressure is at the top of the tree
because water is being lost at the top of the tree
this sets up a tension which is greatest at the top of the tree
there is a, hydrostatic pressure/tension, gradient in the xylem vessels
some pressure is (inevitably) lost on the way down the tree

9 transpiration/loss of water vapour/loss of water by evaporation, from the leaves occurs during the day
because the stomata are open
this results in tension in the xylem (vessels)
walls of xylem vessels are pulled slightly inwards/vessels shrink slightly
overall eff ect is for diameter of whole trunk to, shrink/get smaller
stomata close at night, so no transpiration at night


11 a the loss of water vapour
from the leaves/from the surface of a plant [2]
b light intensity temperature [2]
c rate of water uptake shows the same pattern as rate of transpiration AW
but there is a time delay, with changes in rate of transpiration occurring before changes in water uptake AW [2]
d transpiration causes water uptake
loss of water (by transpiration) sets up a water potential gradient in the plant
water potential in roots is lower than water potential in soil
therefore water enters plant through roots
time delay between rate of transpiration and rate of water uptake is due to time taken for effect of transpiration to be transmitted through the plant AW [max. 4]

12 a thick cuticle (on lower epidermis/outer surface when rolled)
leaf rolled up (due to activity of hinge cells)
hairy upper epidermis/leaf is hairy
stomata absent from lower epidermis/stomata only present in upper epidermis
sunken stomata/stomata in pits/stomata in grooves (in upper epidermis) [max. 3]

b thick cuticle:
cuticle contains a (fatty and relatively) waterproof substance called cutin
the thicker it is, the more eff ective

leaf rolled up:
encloses a humid atmosphere/allows a humid atmosphere to build up

hairy:
hairs trap a layer of (still) moist air next to the leaf

stomata absent from lower epidermis:
reduces/prevents, transpiration from, lower epidermis/exposed surface
sunken stomata:
allows a humid (still) atmosphere to build up around the stomata

Allow 1 mark on one occasion only for ‘reduces the steepness of the water potential gradient from leaf to air inside the (rolled) leaf’ if relevant [max. 6]

13
a hydrogen ions are actively transported out of the, sieve element/companion cell [1]

b there are more hydrogen ions/there is a build-up of hydrogen ions, outside the sieve element–companion cell units compared with inside
hydrogen ions are positively charged [2]

c ATP is needed for the active transport of hydrogen ions out of the tubes [1]

14 a actual length = observed length/magnifi cation,
A = I:M
observed length of sieve element = 51 mm (allow ۫ mm)
actual length = 51 mm/150 = 0.51 mm accept
conversion of mm to μm: answer = 510 μm [3]

b i 1 metre = 1000 mm
1000/0.51 = 1961(to nearest whole number)
ili
1 metre = 1 000 000 μm
1 000 000/510 = 1961 (to nearest whole number) [2]

ii to maintain the pressure gradient inside the sieve tubes
without the sieve plates the diff erent pressures at source and sink would quickly equilibrate [max. 1]

iii sieve pores [1]

c (sieve element is 0.51 mm long)
(1 hour = 3600 seconds)
3600 seconds to travel 1 metre
therefore:
0.51/1000 × 3600 seconds to travel 0.51 mm
= 1.8 seconds (to one decimal place)
Accept 510 μm and 1 000 000 (μm) instead of 0.51 mm and 1000 (mm). [3]


Zaključak

Root system is the first organ in contact with the different components of the soil and water. By their exudates and their effects on rhizosphere activities (proliferation of microorganisms, metal chelation, acidification, etc.) plant roots can tolerate and in some cases accumulate high levels of Pb. An overall higher rate of accumulation was observed in roots rather than leaves in several species. Almost 90% of Pb accumulated in a number of species of the Brassicaceae family (Kumar et al., 1995) and some crops species such as Z. mays (Małkowski et al., 2002) and Pistia stratiotes (Vesely et al., 2012) was located in roots. This accumulator potential can be used in phytoremediation process. Rhizofiltration is a subset technique that uses both terrestrial and aquatic plants roots to absorb, concentrate, and precipitate metals from polluted water to their biomass (Dushenkov et al., 1995). This technique is cost-effective, and can be used for site restoration including maintenance of the biological activities of the polluted site. In this context, several plants have been identified whose roots could be used to clean up land contaminated by Pb. Therefore, improvement of the capacity of plant roots to tolerate and accumulate Pb by genetic engineering should open up new opportunities for rhizoremediation.


Groundwater Pollution

Plants cannot absorb all the excess nitrogen in the soil. Those extra nitrogen levels slowly leach out of the soil through water runoff the nitrogen is effectively in the form of nitrates due to microbial conversion when it leaches from the soil. As a result, groundwater and drinking water become contaminated from the nitrate levels. Between harming the plants and the surrounding water supplies, high nitrogen levels around plants need to be closely monitored and amended for natural harmony.

Writing professionally since 2010, Amy Rodriguez cultivates successful cacti, succulents, bulbs, carnivorous plants and orchids at home. With an electronics degree and more than 10 years of experience, she applies her love of gadgets to the gardening world as she continues her education through college classes and gardening activities.


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