Molecular basis of phosphoinositide-specific phospholipase C signaling pathways in plant cells

In plants external stimulus can be perceived and amplified in the cells by functional signaling cascades. Phosphoinositide-specific phospholipase C is an enzyme shown to initiate and provide key events in the cellular responses to extracellular signals. Both substrate and products of phospholipase C are involved in the regulation of numerous processes in plant cells. In this review, we focused on molecular basis of the phosphoinositide-specific phospholipase C signaling pathways. The data analyzed will help to elucidate the mechanisms responsible for plant’s ability to respond to a variety of biotic and abiotic stress signals.

In tro duc tion.Phosphoinositide-spe cific PLC (PLC) is a key en zyme of the phosphoinositide sig nal transduction in cells of bac te ria, pro to zoa, plants and an i mals.It hy dro ly ses phosphatidylinositol 4, 5-biphosphate pro duc ing inositol trisphosphate and diacylglycerol [1].In the plant PLC plays an im por tant role in the di ver sity of phys i o log i cal pro cesses.It is acti vated in re sponse to var i ous en vi ron men tal effectors such as salt [2,3], cold [4,5], os motic stresses and drought.More over, the plant PLC is a com po nent of sig nal ing path ways of some phytohormones, for ex ample, abscisic acid (ABA) [10,11] and cytokinin [12].
Hav ing the unique func tions, both sub strate and PLC prod ucts par tic i pate in the reg u la tion of nu mer ous pro cesses [1,13].Inositol trisphosphate and hexakisphosphate (inositol trisphosphate trans mu ta tion prod uct) cause Ca 2+ re lease from the intracellular storage [14,15].Diacylglycerol can be phosphorylated by the diacylglycerol kinase pro duc ing the lipid mes senger, phos pha tid ic acid.The ves i cle trans port in a cell is shown to be as so ci ated closely with intracellular lo caliza tion, cyclization, mo tion and deg ra da tion of proteins, and reg u lated by the phospholipid mol e cules includ ing phosphatidylinositol 4, 5-biphosphate and phos pha tid ic acid [16, 1 7].
Study on PLC ac tiv ity both in vi tro and in vivo is com pli cated by the phosphatidylinositol 4, 5-biphosphate low con tent in the higher plant or ganisms, and reg is tra tion of inositol trisphosphate with thin layer chro ma tog ra phy is sel dom suc cess ful [18].The inositol trisphosphate anal y sis is of ten per formed with the Amersham TRK1000 test [19].In ves ti ga tion of PLC sig nal ing path ways is mostly based on ap pli ca tion of the en zyme in hib i tors in clud ing U73122 and its in active an a log U73343.How ever, ac cord ing to some infor ma tion, un der the in flu ence of U73122, the slow increas ing of Ca 2+ in a Ca 2+ -free me dium as well as blocking Ca 2+ chan nels of L-type are ob served [21].PLC is also stud ied us ing trans gen ic plants.The ex per i ments on stim u la tion or de pres sion of the PLC gene ex pression, re quired to clar ify the PLC func tions in the plant or gan isms, have been much suc cess ful [22][23][24].The study on PLC char ac ter is tics will as sist in fur ther compre hen sion of the mech a nisms of phospholipid sig naling path ways con sid er ing dif fer ent as pects of plant growth and de vel op ment.
PLC Mo lec u lar Struc ture.The five types (b, g, d, e and f) are dis tin guished among the mam ma lian PLC.They con tain PH-do main that is a ba sic se quence required for the at tach ment to plasma mem brane, binding with sub strate and ca tal y sis.They also have EF-do main im por tant for the en zyme ac ti va tion, Xand Y-se quences rep re sent ing the cat a lytic cen tre, and C2-do main en sur ing in ter ac tion with Ca 2+ and lipids [25,26].Such struc ture is typ i cal for PLC d-isoform while there are ad di tional do mains in other PLC fam ilies.The plant PLC is struc tur ally iden ti cal to the small est mam ma lian PLC z-isoform of about 60-70 kD, which has two EF-hand se quences, X-, Y-, and C2-do mains but not the PH-do main typ i cal for the other PLC isoforms [27].
How ever, it should be noted that on the se quence level PLC is closer to the mam ma lian PLC d-isoform [28,29].The plant PLC does not con tain PH-do main, which is nec es sary for the in ter ac tion of the an i mal PLC with plasma mem branes.PLC con tains C2-Ca 2+ /phospholipids -bind ing do main fix ing the cata lytic cen tre in cor rect po si tion.This do main par tic ipates in the in ter ac tion with mem brane but its pres ence is not suf fi cient for the en zyme to form cat a lyt i cally active state and other PLC do mains are re quired to associate with plasma membranes [26,30].
PLC Genes Ex pres sion and Evo lu tion.For the first time, DNA en cod ing for PLC was iden ti fied in an imal cells in 1988 [31].Seven years later the Arabidopsis thaliana L. [2] and soy bean (Glycine max L.) PLCs were cloned and the pres ence of this en zyme pro tein was dem on strated in both plasma mem brane and cytosol [32].Now a days, a lot of the ac tive PLC cod ing genes are known and the PLC pro teins were obtained from cer tain plants, for ex am ple, po tato (Solanum tuberosum L.) [33], mungbean (Vigna radiata L.), maise (Zea mays L.), rice (Oryza sativa L.) [1], and pe tu nia (Pe tu nia inflata L.) [30].
In the A. thaliana ge nome there are 9 AtPLC genes [22,29].AtPLC1 -AtPLC5 genes en code for PLC the activ ity of which was proved in vi tro, whereas the en zymatic ac tiv ity of AtPLC8 and AtPLC9 prod ucts is hardly prob a ble [22,34].AtPLC6 and AtPLC7 con tain the domain re quired for the PLC ac tiv ity and en code the ac tive en zyme.Pre vi ously it was es tab lished that AtPLC7 encodes in com plete and non-func tional pro tein with molec u lar mass of 30 KD [22].How ever, sub se quently there was dis cov ered the full-chain AtPLC7 DNA, proba bly en cod ing the PLC func tional pro tein [35].
The cat a lytic do mains of all PLCs have the sim i lar struc ture and ox i da tion-re duc tion ca tal y sis mech a nism.The con ser va tism of to pol ogy and el e ments of the active site in di cate their or i gin from a sin gle al lied protein.The phylo gen etic his tory of eukaryotic PLC proba bly in cludes sev eral stages of chain elon ga tion and gene du pli ca tion.The AtPLC gene fam ily evo lu tion seems to go through sev eral phases.The gene tan dem of AtPLC8 and AtPLC9 arose due to the lo cal du pli ca tion on later stages.AtPLC1, AtPLC4 and AtPLC5 make up a group of genes com bined from small DNA frag ments.Prob a bly, the pre cur sor of AtPLC4/AtPLC5 and AtPLC1/AtPLC3 arose from a sin gle gene dur ing the du pli ca tion in the 5 th chro mo some with the fur ther AtPLC3 du pli ca tion and translocation onto the 4 th chromosome responsible for the further gene distribution [35].
The ex pres sion of AtPLC gene in creases in response to such en vi ron men tal fac tors as de hy dra tion, salinization and cold stress [2,36].The po tato PLC genes are ex pressed in re sponse to the wound ing stress and wa ter deficit [33].
The AtPLC1 and AtPLC6 tran scrip tion is in duced in re sponse to some abiotical stresses in clud ing de hydra tion, sa lin ity and cold stress [2,34] (see the ta ble).The tran scrip tion level in cre ment is sup posed to re sult in the AtPLC pro tein in crease and thereby it ac ti vates the sig nal ing path ways of up-or down-reg u la tion of genes par tic i pat ing in var i ous cell re ac tions.For ex am -ple, the AtPLC1 tran scrip tion, the en zyme ac ti va tion and in crease in the inositol trisphosphate level were dem on strated to be re quired for the fur ther gene ex pression in response to the abscisic acid influence.
AtPLC1 is ac ti vated in re sponse to the in flu ence of salt, abscisic acid, cold and de hy dra tion.The AtPLC2 gene ex pres sion is not in duced un der the in flu ence of abiotical stresses [37].Un like the AtPLC2, the other 8 genes of AtPLC are in duced in re sponse to the ex ter nal in flu ence.For AtPLC8 and AtPLC9 the tran scrip tion level in creases less than two fold un der the in flu ence of all ex ter nal stim uli [35].(More than two-fold in ten si fica tion of) The AtPLC6 in duc tion rise more than two -fold was dem on strated as a re sult of ac tion of some exter nal fac tors [2].
The phys i o log i cal role of PLC in the re sponse of plants to the abiotic stress was de scribed us ing the trans gen ic maize.The abiotic stress does not change either the wild phe no type or the phe no type of plants with de pressed or height ened PLC gene ex pres sion which were grown in the op ti mal con di tions.How ever, un der the in flu ence of wa ter stress the plants with di min ished PLC1 ex pres sion dif fered by a low wa ter con tent in tissues, osmoregulation de te ri o ra tion, photosynthetic activ ity de cay, high per cent age of ion loss, higher lipid peroxidation in ten sity and lesser pro duc tiv ity in com - par i son with the wild.There was drawn a con clu sion on fail ure of sig nal transduction mech a nism and, be cause of this, in abil ity of cells of pro vid ing the ad ap ta tion to the stress which in di cates the es sen tial role of PLC genes in the regulation of response to the stress caused by water deficit [38].PLC Ki netic Prop er ties.Most of the ac tive plant's PLCs were ob tained from cytosol, but as the sub strate of this en zyme is as so ci ated with mem brane the en zyme ac tiv ity in cytosol and mem branes may dif fer.Be cause of this one of the most in ter est ing tasks is to study the ac ti va tion of PLC on the mem brane sur face.The PLC ac tiv ity in Catharanthus roseus L was de scribed us ing the lipid sub strates, dis trib uted in the phospholipid vesi cles, phospholipid mi celles and monolayer in the air-wa ter me dium [39].The ap pli ca tion of P 33 -la beled sub strate for the di rect mea sure ment of the Catharanthus roseus L. PLC ac tiv ity re vealed de pendence of the monolayer PC-PS pro por tion and amount on the pres sure.The PLC ac tiv ity in creases when the pres sure is raised up to 20 mN/m 2 and reaches the max imum un der these con di tions.The fur ther pres sur iza tion leads to the PLÑ ac tiv ity de cay.Prob a bly, the PLC activ ity re duc tion is caused by de crease in the en zyme abil ity to bind the sub strate.This phe nom e non is specific and seems to de pend on the phosphatidylinositol 4, 5-biphosphate con cen tra tion.These re sults dif fer from those re ceived for the an i mal d-PLC and are sim ilar to b-PLC.This is un ex pected be cause struc tur ally the plant PLC is closer to the d-PLC isoform.However, it was dem on strated that the d-PLC isoform activ ity de creases lin early at in creas ing sur face pres sure.What is the rea son of the dif fer ences be tween the activ i ties of the plant PLC and d-PLC in a monolayer is un known, but the de pend ence of dif fer ent isoforms ac tiv ity on the char ac ter is tics of the sur faces which they in ter act with was de ter mined.Thus, as a re sult of study ing the PLC of plants and other or gan isms [40,41] it was es tab lished that the phosphatidylinositol 4,5-biphosphate hy dro ly sis in a monolayer de pended on the sur face pres sure de ter min ing the pe cu liar i ties of in ter ac tion be tween the en zymes of this fam ily and the lipid sur face [39].
This in for ma tion and the re sults, ob tained us ing the monolayer sub strate where the PLC ac tiv ity de creased as the sub strate pres sure in creased, in di cate that PLC has to pen e trate through the lipid ag gre gates in the case of the sub strates bind ing and hy dro ly sis.The data on ve sic u lar bind ing con firms the plant PLC in ter ac tion with the mem brane sur face by the phosphatidylinositol 4, 5-biphosphate sub strate.To pro vide the for ma tion of the PLC-me di ated sec ond ary mes sen gers PLC can inter act with the mem brane sur face in spe cific non-cat alytic way with fur ther bind ing or sur face re or ga ni zation.This can pro mote a sta ble fix a tion of PLC on the mem brane sur face [42].
The un ex pected re sults were re ceived dur ing in vesti ga tion of the sur face in ter act ing site and fur ther binding of the lipid sub strate in side the mem brane.Prob ably, the Mi chae lis-Menten equa tion curve with Hill coef fi cient close to 1 in di cates the ex is tence of a sin gle bind ing site.The an i mal PLC amino-ter mi nal re gion, con tain ing pleckstrin ho mol o gous do main, is nec es sary for bind ing with phospholipid ves i cles which con tain phosphatidylinositol 4, 5-biphosphate.These re sults con firm that the pres ence of phosphatidylinositol 4, 5-biphosphate can be an im por tant fac tor, re quired for the lo cal iza tion of the pro teins, which con tain this domain on the mem brane sur face.How ever, the pleckstrin ho mol o gous do main has not been re vealed among the prod ucts of the plant PLC genes.Per haps, the plant PLC ini tially has to af fil i ate with phosphatidylinositol 4, 5-biphosphate in the catalytic site n [43].
The en an tio mer pure an a logues of all nat u ral PLC sub strates, in clud ing both long-and short-chain phosphatidylinositol 4, 5-biphosphates, phos pha tid ylinositol 5-phos phates and non-phosphorylated phosphatidylinositols, were syn the sised.The sub strates phosphorylated at the 4-inositol bond (phosphatidylinositol 4, 5-biphosphate and phosphatidylinositol 5-phos phate) have very sim i lar kinetic prop er ties and their phosphorylation pro gresses 20-30 times more ac tively than those non-phosphorylated (phosphatidylinositol 4-phos phate and phosphoinositide).So it can be con cluded that the in terac tion ex actly with the 4-phos phate group is re quired for the PLC ca tal y sis.Fur ther more, the bind ing af fin ity of all four groups is rather sim i lar that in di cates the energy suf fi ciency of en zy matic bind ing with the 4-phosphate group for the catalysis.
The spec i fic ity of the wheat root PLC to wards the polyphosphoinositides de pends on the var i ous fac tors: pH (pH 6-7 -the phosphoinositide-phos phate hy dro lysis, pH 6-6.5 -phosphoinositide 4, 5-biphosphate hydro ly sis) and (kind of the) ions.The cal cium, man ganese and co balt ions at the con cen tra tion of 4 mM dimin ish the PLC spec i fic ity to wards phosphoinositide-biphosphate and in ten sify the phosphoinositide-phos phate hy dro ly sis.At high calcium con cen tra tion the PLC spec i fic ity changes in such row: phosphoinositide > phosphoinositide-4-phos phate > phosphoinositide 4, 5-biphosphate [45].
PLC can be di vided into 2 groups by the in vi tro prop er ties: sol u ble forms which are spe cific to wards phosphoinositide and re quire cal cium of mM con cen trations for ca tal y sis, and PLCs, at tached to the plasmalemma with a sub strate spec i fic ity to wards the polyphosphoinositides, re quir ing µM con cen tra tions of cal cium for ac ti va tion (the op ti mum is 0.1-10 µM) [30].The pu ri fied mem brane PLC is highly spe cific to wards phos phates (100% ac tiv ity); crude en zyme is more specific to wards the polyphosphoinositides (10% ac tiv ity for phosphoinositide-phos phate and 30% for phosphoinositide-4, 5-biphosphate).The ac tiv ity of pu ri fied form to wards phosphoinositide 4, 5-biphosphate is re stored by ad di tion of a pro tein lost dur ing the en zyme pu ri fi ca tion.How ever, it was dem onstrated that this reg u la tory fac tor is not a G-pro tein [45].
Reg u la tion of PLC ac tiv ity.G-pro teins.Heterotrimeric G-pro tein con sists of a-, b-and g-subunits and op er ates as a mo lec u lar switch con trol ling a great num ber of cel lu lar re ac tions, trans mit ting the signal from the cell sur face re cep tors to the intracellular eliñitors, such as PLC, phospholipase D, cyclases, ion chan nels, phosphodiesterases, etc. [46].In the hu man ge nome there are about 1000 genes for G-pro teins, whereas in the A. thaliana ge nome only one gene of G-pro tein, par tic i pat ing in the cell cy cle reg u la tion [47] and abscisic acid sig nal ing in the guard cells [48], has been found up to now.A G-pro tein was dem on strated to par tic i pate in the ion chan nel reg u la tion [49] and cellu lar pro lif er a tion of the A. thaliana plants [50].Moreover, the mu ta tions in the A. thaliana and rice G-protein re sult in the dam age of a wide range of pro cesses, such as seed germination, spear and root growth, stoma movement [50].
Three le gumes G-pro tein sub units were ob tained and de scribed; G-pro tein par tic i pa tion in the salt and ther mal stress sig nal transduction, and the in ter ac tion be tween G-pro tein and PLC were dem on strated.The in ter ac tion be tween the G-pro tein a-sub unit is an impor tant stage of salinization sig nal transduction.The thermotolerance is based on the Gb-me di ated sig nal transduction.A fur ther study is re quired to clar ify the reg u la tion of these pro teins in re sponse to the salt and ther mal stresses, and to un der stand their role in the adaptation [51].
The change in inositol trisphosphate con cen tra tion is ob served in re sponse to an in crease in the ty ro sine kinase ac tiv ity and PLC ac ti va tion dur ing the G-pro tein ac ti va tion at in fec tion of Cit rus limon L. le sion with Altemaria al ter nate fun gus.It was es tab lished that two sig nal path ways are ac ti vated: one -with par tic i pa tion of G-pro tein, an other -with ty ro sine kinase-de pend ent PLC.A pos si bil ity of the re la tion be tween PLC ac ti vation and ty ro sine kinases requires fur ther research [52].
The reg u la tion of PLC ac tiv ity by cal cium ions.Cal cium is a main ac ti va tor among the ions able to influ ence the PLC ac tiv ity.The op ti mum cal cium concen tra tion for the PLC ac tiv ity in microsome frac tions and plasma mem branes of Bras sica napus cells is 10 -5 -10 -4 M [53].
The max i mum phosphoinositide 4, 5-biphosphate hy dro ly sis by the soya PLC is ob served at pH op ti mum of 6.5-7.5 [54].The lipid hy dro ly sis by the po tato PLC in ten si fied at the cal cium con cen tra tion of 100 µM.The spec i fic ity of PLC 1 to wards phosphoinositide 4, 5-biphosphate starts to di min ish at more than 100 µM cal cium, whereas PLC 2 and PLC 3 lose the spec i fic ity at a higher cal cium con cen tra tion of 100-10000 µM [33].
The A. thaliana PLC 1 in hi bi tion does not de press the Cor and RD29a stress genes ex pres sion in duced by the abscisic acid [20].Per haps, the syn the sis of the cyclic ADP-ribose will me di ate the ini tial cytosolic calcium con cen tra tion rise re sult ing in the PLC ac ti va tion.How ever, this does not rule out the pos si bil ity that other isoforms of PLC can ini ti ate si mul ta neously a pri mary re sponse and a cal cium flux into the cell even at low cal cium con cen tra tions.At least one of the A. thaliana PLC isoforms, namely AtPLC4, is ac tive in the absence of calcium [22].
AtPLC1 mainly hy dro ly ses phosphoinositide 4,5-biphosphate.At the op ti mum cal cium con cen tration phosphoinositide 4, 5-biphosphate is hy dro lyzed by 100 times more ef fi ciently than phosphoinositide.The max i mum hy dro ly sis is ob served at 1-50 µM calcium, and at the cal cium con cen tra tion of more than 1 mM the phosphoinositide hy dro ly sis pre vails (like the po tato and soya PLC).The Mi chae lis-Menten con stant for the PLC of C. roses roots is 0.0518 and the sub strate con stant is 45.5µM, the max i mum re ac tion rate is 137.2 picomoles/min [39].
A need for Ca 2+ for the ac ti va tion of many plant PLC seem to in di cate that inositol trisphosphate for mation is not a pri mary re sponse to the stress since the Ca 2+ level rise is first re quired for the PLC ac ti va tion and phosphoinositide for ma tion.The cal cium level can increase due to the cal cium flux from ex tra cel lu lar medium or be cause of the ac tion of sec ond ary mes sen gers able to re lease Ca 2+ , e.g.cy clic ADP-ribose [56,57], nic o tinic acid adeninedinucleotidphosphate [58], sphingosin-1-phos phate [59], hy dro gen per ox ide [60], and hexakisphosphate [61].
The study on reg u la tion of five AtPLC isoforms by cal cium dem on strated that A. thaliana PLC 2 is the most sus cep ti ble to the cal cium ion ef fect: at the concen tra tion of 10 nM its ac tiv ity was 80% of max i mum.The sus cep ti bil ity to the µM cal cium con cen tra tions dimin ishes in the fol low ing or der: PLC4 -PLC5 -PLC1.PLC 3 has the weak est re sponse to cal cium: at 10 nM con cen tra tion the ac tiv ity is 15% of max i mum.PLC4 is the most sta ble at low cal cium level -its ac tiv ity is 20% of max i mum in the pres ence of EGTA.All A. thaliana PLCs ex cept for PLC 3 are the most ac tive at the calcium con cen tra tion of 3 µM and pre serve their ac tiv ity on the same level at the cal cium con cen tra tion of 10 µM.In case of mi cro mo lar cal cium con cen tra tions the PLC isoform ac tiv i ties dif fer.PLC 2, PLC 4 and PLC 5 reach the max i mum ac tiv ity level at the cal cium concen tra tion of 1µM whereas PLC1 and PLC3 re quire higher cal cium con cen tra tions for the max i mum ac tivity.The over lap ping of the A. thaliana var i ous PLC genes ex pres sion in di cates their func tional ex cess.How ever, it is also pos si ble that the reg u la tion of each gene and PLC is ex erted in dif fer ent ways.The cal cium re lease un der the in flu ence of one of the PLC isoforms can cause an ac ti va tion of the oth ers.The per ma nent expres sion of A. thaliana PLC2 and PLC3 ev i dences for their par tic i pa tion in the pri mary re sponse to a stress, caus ing the cal cium ion concentration rise to induce the PLC1, PLC4 and PLC4 genes expression along with other genes ac ti vated by calcium [22].
The Mo lec u lar Mech a nisms of PLC Sig nal Pathway Re al iza tion in the Plant Cells.Inositol triphosphate, hexakisphosphate.The inositol trisphosphate ad min is tra tion into the cells re sults in the cy to plas mic cal cium con cen tra tion rise, stomata closing, proto plasts swell ing, fer til iza tion tube growth in hibi tion, and plasmodesma clos ing [62].Inositol trisphosphate is a sec ond ary mes sen ger which re lease cal cium out of the intracellular de pot in plant cells [14].High-af fin ity sites for inositol trisphosphate bind ing in an i mals are lo cated on the endoplasmic re tic u lum (ER).An as sumed inositol trisphosphate re cep tor was obtained from the Vigna ra di ate L and char ac ter ised.In com par i son with the an i mal cell re cep tors it is smaller (110 against 250 kD), and its ac tiv ity is in ten si fied by the inositol phos phate me tab o lites [63].The ex is tence of the cal cium chan nels sen si tive to inositol trisphosphate on the non-vacuolar mem branes was proved.How ever, up to now the at tempt to ex tract the inositol triphosphate-reg u lated chan nel pro tein has failed.The inositol trisphosphate re cep tors genes homol o gous to the an i mal ones also have not been found in the A. thaliana ge nome [64].In the plants Chenopodium rubrum L. the inositol trisphosphate bind ing sites are lo cated on the ER [54].It was dem onstrated that the inositide-2, 4, 5-trisphosphate stim ulates cal cium re lease from the intracellular de pot rather ef fi ciently, but slighter than inositide-1, 4, 5-trisphosphate [65].Per haps, the inositol triphosphate-phytase com plex in ter acts with the inositol trisphosphate re cep tors re sult ing in cal cium release from the microsome frac tions whereas inotitide-1,3,4-trisphosphate does not have such ca pabil ity since it does not bind to the re cep tors [65].Study in vi tro on the Chenopodium Rubrum L .plants re vealed the in ter ac tion of inositol triphosphate-phytase with the inositol trisphosphate re cep tors, which re quires the nanomolar con cen tra tion of the sub strate.The inositol trisphosphate bind ing with a highly af fine non-cat a lytic site of the phytase re sults in the es sen tial con for ma tion changes in the en zyme.This causes the for ma tion of inositol triphosphate-phytase-re cep tor com plex with much more ac tive re lease of cal cium com par ing to free inositol trisphosphate [63].These in ves ti ga tions are in fa vour of the ex is tence of a new signal cascade, regulating the calcium homeostasis, which is mediated by the hexakisphosphate-phytase system in the plant cells [65].
A role of hexakisphosphate is not stud ied enough.The S. tuberosum L. and V. faba L. stomata cells treatment with abscisic acid in creases the level.The hexakisphosphate was shown to screen the in hib it ing ef fect of abscisic acid and cal cium on the po tas sium chan nels [15].The hexakisphosphate and phos pha tid ic acid, formed in the yeast from inositol trisphosphate and diacylglycerol, reg u late the gene tran scrip tion and mRNA trans port [66].This ex plains the ab sence of the genes, cod ing the cal cium chan nels which are reg u lated by inositol triphosphate, and the ab sence of the pro tein kinase C genes in the genome of these organisms.
Diacylglycerol, diacylglcerolpyrophosphate, phospha tid ic acid.The in crease in DAG level stim u lates the H + -ATP-ac tiv ity and the stomata open ing, af fects cell di vi sion, ham pers the pro tein move ment through the plasmodesma and in ten si fies the phosphorylation [67].The cell treat ment with DAG in vivo re sults in the change of cytoskeleton flex i bil ity the re duc tion of intervacuolar fil a ment ten sion.DAG is also in volved into the pro cess of mi to sis in the cells of sta men fil aments.Fur ther more, DAG in duces the ion ab sorp tion in the iso lated proto plasts of guard cells and stomata open ing [62].How ever, it is not es tab lished whether these ef fects are caused di rectly by DAG or its me tab olites (for ex am ple, fatty ac ids and phos pha tid ic acid (PA)).Most of the ob ser va tions in di cate the DAG phosphorylation im me di ately af ter its for ma tion [62, 69, and 70].
The phos pha tid ic acid rise was dis cov ered in var ious types of the plant cells un der the in flu ence of the osmotic stress, wound, patho gens, abscisic acid, ox i dative stress, Nod-fac tors, and drought [70].The for ma -tion of 18:3/16:3-PA through the DAG phosphorylated by the DAG kinase was ob served un der the freez ing and in flu ence of the patho gens.The phospholipase D is also an im por tant PA generator [1,24].
The PA sig nal ling is al ways im pet u ous and transient that is en sured by the sig nal de pres sion mech anism [70].The res to ra tion of the ini tial con cen tra tion is very im por tant for most of the sig nal mol e cules.In the plant cells the PA sig nal ling weak ens due to its phosphorylation by PA kinase with pro duc ing diacylglcerolpyrophosphate (DGPF) [62,70].At rest the DGPF con cen tra tion in cells is very low, and the ex pres sion level of PA kinase, which is re spon si ble for DGFP for ma tion, re mains con stant for all plants.This stip u lates a close as so ci a tion be tween the DGPF forma tion and its pre cur sor PA avail abil ity.Since the DGPF for ma tion co in cides with PA level low er ing, the PA kinase can take part in the PA sig nals de pression.In vivo the PA kinase ac ti va tion was dis cov ered in yeast and in many plant sys tems in re sponse to the var i ous phys i o log i cal stim uli, in clud ing his hy per osmotic stress, drought and patho gens at tack.Thus, a pos si ble role of DGPF as a sec ond ary me di a tor for PLC sig nal trans-duction in the plant cells should not be ex cluded [70].
The Phosphatidylinositol 4, 5-biphosphate Func tions in the Plant Cells.In the an i mal cells phosphatidylinositol 4, 5-biphosphate is not only a substrate of PLC, but it can be also a sig nal ing mol e cule influ enc ing var i ous bi o log i cal pro cesses in cells [13].The phosphatidylinositol 4, 5-biphosphate of higher plants is a reg u la tor of the sig nal path ways.The change in intracellular phosphatidylinositol 4, 5-biphosphate level is ob served in re sponse to the in flu ence of light, cold, grav i ta tional stim u la tion, ox i da tive stress, G-pro teins ac -ti va tion, and patho genic elicitors [74].Pos si ble processes with phosphatidylinositol 4, 5-biphosphate as sistance are listed here un der: 1. Ion chan nels reg u la tion.Phosphatidylinositol 4, 5-biphosphate is con sid ered to be a key reg u la tor of ion chan nels ac tiv ity, es pe cially K + [75].The phosphatidylinositol 4, 5-biphosphate ac cu mu la tion in the plant plasma mem brane in duced by the salt stress can re flect this func tion [13].
2. Mem branes for ma tion and flu id ity.Dur ing the mam ma lian cell di vi sion phosphatidylinositol 4, 5-biphosphate is mainly lo cal ized in the mem brane and is re quired for the cytokinesis ac com plish ment for the ac tive area for ma tion, ef fec tive mem brane fu sion, and cell division [76,77].
3. The cytoskeleton re or gani sa tion.Phosphatidylinositol 4, 5-biphosphate is in volved into the cleav age fur row for ma tion.It is bound with actin-reg u la tory pro teins and can in flu ence their ac tivity [78, 79, and 80].More over, an in crease in the lo cal phosphatidylinositol 4, 5-biphosphate level can promote pro teins in ter ac tion with the plasma mem brane hav ing spe cific phosphatidylinositol 4,5-biphosphatebind ing sites [79,81].The plants are sup posed to contain sev eral such do mains in clud ing the cytoskeleton or gan is ing pro teins [1,13].The low phos pha tid ylinositol 4, 5-biphosphate level in plants can be explained by ei ther a higher af fin ity of the plant pro tein do mains to wards lipids com pared with the mam malian ones, or the af fin ity of com ple men tary pro tein domains to wards the other com po nents, which are nec essary for ef fi cient bind ing with the plasma mem brane.The con fir ma tion of such func tions may be the response ob served dur ing cell di vi sion and salt stress as well as changes in the cy to plas mic re tic u lum, in duced by U71322, and an in flu ence on the vac u oles morphol ogy [13].The phosphatidylinositol 4, 5-biphosphate par tic i pa tion in actin cytoskeleton regu la tion was also dem on strated dur ing the fer til iza tion tube growth [30].
4. The mem brane trans por ta tion.In the mam ma lian cells a lot of polyphosphoinositides are in volved into the exocytosis and/or endocytosis [79,82].How ever, the phosphatidylinositol 4, 5-biphosphate ac cu mu lation in small ves i cle struc tures is not ob served [84].The phosphatidylinositol 4, 5-biphosphate po lar gra di ent can re flect the mem brane trans por ta tion events dur ing the root fi brils growth [30,84].
De spite the va ri ety of PLC sig nal ling mech a nisms, a mi nor quan tity of phosphatidylinositol 4, 5-biphosphate pres ent in the mem branes of higher plants is reg is tered on the re cently formed mem branes of the di vid ing cells, ac cu mu lat ing in the mem branes in re sponse to the salt stress [13].The de crease in phosphatidylinositol 4, 5-biphosphate level in the stomata guard cells in re sponse to the abscisic acid influ ence in di cates its par tic i pa tion in the abscisic acid sig nal ling cas cades, ac ti vat ing the stomata clos ing [55].It was dem on strated that the phosphatidylinositol 4,5-biphosphate-bind ing pro tein in hib its the stomata open ing in duced by light, re sult ing in re duc tion of the free phosphatidylinositol 4,5-biphosphate level decrease in the inositol trisphosphate and phos pha tid ic acid for ma tion un der the in flu ence of PLC and D; besides, it stops the stomata clos ing caused by abscisic acid [74].
Light-de pend ent in cre ment of phosphatidylinositol 4,5-biphosphate con tent in the plasma mem brane can oc cur not only due to in creas ing syn the sis but also be cause of re duc tion of phosphatidylinositol 4,5-biphosphate hy dro ly sis by PLC.The di min ished PLC ex pres sion re duces the in hibi tory ef fect of abscisic acid on the seed ger mi na tion and the in flu ence of this phytohormone on the ex pres sion of the drought-and cold-re spon sive genes [37].
PLC plays a sig nif i cant role in the abscisic acid signal transduction in the stomata guard cells.U73122 (a PLC in hib i tor) re duces the guard cells re sponse to the abscisic acid ac tion and Ca 2+ fluc tu a tions in cytosol [11].More over, a de crease in the PLC level in the stomata guard cells par tially pre vents the in hi bi tion of stomata open ing by abscisic acid [22,23].U73122 was used to study PLC in the light-in duced stomata open ing .The stomata guard cells af ter the in hib i tor treat ment speed up the open ing caused by cir ca dian rhythm.The ques tion whether the PLC reg u la tion of the re sponse to the light in flu ence oc curs in the same way as the abscisic acid sig nal ling, still re mains open [74].
Thus, de spite the re cent sig nif i cant achieve ments in the study on the PLC ki net ics, gene ex pres sion and partic i pa tion of this en zyme in the cell sig nal cas cades, the mech a nisms of reali sa tion of PLC-me di ated sig nal ling path way re quire fur ther in ten sive many-sided research.
The work was sup ported by the Ukrai nian Foun dation for Fun da men tal re search, grant F14.4/253-2007.
Enhancement of phosphatydylinositol specific phospholipase C expression in plant cells in response to abiotic factors and influence of abscisic acid.