Molecular and genetic approaches for investigation of phospholipase D role in plant cells

The cognition of the nature of intracellular signaling and its role in the regulation of cell metabolism is one of intensively developing trends of modern biology. The results of investigations on signaling systems of plant cells testify to the fact that phospholipids (PL) are not just structural components of membranes, but also precursors of second messengers of intracellular signaling. Many stresses and a number of phytohormones cause sharp increase in the content of phosphatidic acid (PA) in plant cells which proves activation of phospholipases, in particular, phospholipase D, (PLD, EC 3.1.4.4). PLD is an enzyme, wide-spread in both plant and animal kingdoms, which hydrolyzes structural PL via terminal phosphodiester bond with subsequent formation of PA and free functional groups [1]. Together with primary alcohols PLD also catalyzes the reaction of transesterification with the formation of phosphatidyl alcohols (phosphatidylbutanol, in particular) [2] – the compounds which are successfully used for studying PLD activity in vivo. Twelve genes of various isoenzymes of PLD – PLDa (3), b (2), g (3), d, e and x (2) [3] were cloned in Arabidopsis thaliana. Seventeen genes of this enzyme were coded in the genome of rice

The cognition of the nature of intracellular signaling and its role in the regulation of cell metabolism is one of intensively developing trends of modern biology.The results of investigations on signaling systems of plant cells testify to the fact that phospholipids (PL) are not just structural components of membranes, but also precursors of second messengers of intracellular signaling.Many stresses and a number of phytohormones cause sharp increase in the content of phosphatidic acid (PA) in plant cells which proves activation of phospholipases, in particular, phospholipase D, (PLD, EC 3.1.4.4).PLD is an enzyme, wide-spread in both plant and animal kingdoms, which hydrolyzes structural PL via terminal phosphodiester bond with subsequent formation of PA and free functional groups [1].Together with primary alcohols PLD also catalyzes the reaction of transesterification with the formation of phosphatidyl alcohols (phosphatidylbutanol, in particular) [2] -the compounds which are successfully used for studying PLD activity in vivo.Twelve genes of various isoenzymes of PLD -PLDa (3), b (2), g (3), d, e and x (2) [3] were cloned in Arabidopsis thaliana.Seventeen genes of this enzyme were coded in the genome of rice -PLDa (8), b (2), d (3), k, x (2) and f [4].The activity of PLD was first registered in 1947 in extracts of carrot roots as an enzyme, hydrolyzing lecitin (phosphatidylcholine) in PL mixtures [5].Later PLD was found in cells of animals, fungi, and bacteria [6].
At present the attention of scientists is focused upon the study of mechanisms of PLD participation in signaling cascades of cells in the process of regulation of cell metabolism of animals and plants [7].PLD plays an important role in many physiological processes, such as germination, growth, senescence of plants and ripening of fruit, as well as in responses to the action of stresses and phytohormones [8,9].
The investigation on the structure of genes and proteins of PLD involves various genetic manipulations with this enzyme which are an essential stage in the determination of its role and functions in cells and plant organism as a whole.An analysis of growth and development of plant lines with PLD deficiency as well as of mutants with T-DNA, inserted in PLD genes, allows defining the significance of various isoenzymes of PLD in the regulation of metabolism in response to the activity of many hormones and stresses.The construction of transgenic plants with simultaneous knockout of genes of several PLD isoenzymes permits to obtain a phenotype, which is more expressed compared to that of mutants with the knockout of a specific gene, and to reveal a PLD function not found before.
Genetic engineering methods in study on PLD role in biological activity of cytokinins and abscisic acid.The study of molecular mechanisms of hormone signal transduction, including the action of cytokinins, is an important part in the investigation of regulation of cell metabolism [10][11][12][13][14][15].Specific bioassays are widely used to reveal possible ways of phytohormone activity.In cotyledons of etiolated seedlings of amaranthus (Amaranthus caudatus L.), exogenously introduced cytokinin 6-benzylaminopurine (BAP) induces rapid accumulation of a red pigment amaranthin, which serves as a basis of specific sensitive and reliable bioassay for the activity of this phytohormone [11,12].The substances which are inhibitors of intracellular signal transmission, decreasing the level of amaranthin biosynthesis, are used to reveal the mechanisms of cytokinin activity in cells.The treatment of plants with primary alcohols (1-butanol, in particular), inhibitors of PA formation, catalyzed by PLD, decreases the level of pigment biosynthesis earlier than transcription blockers.Furthermore, primary alcohols (contrary to the secondary ones) block the accumulation of transcripts of primary response gene to cytokinins [11][12][13].In the cells of periwinkle the stimulating effect of cytokinins on the transcription of primary response genes is also evidently inhibited by primary alcohols.However, their application together with PA results in restoration of induction by cytokinins of the transcription of cytokinins primary response gene [14].The analysis of PL content in corn coleoptiles testifies that the 30-min treatment of sprouts with BAP results in 3-fold increase in concentrations of PA and products of PLD-catalyzed hydrolysis reaction, and in decrease in the amount of phosphatidylethanolamine, the substrate of this enzyme [15].We have demonstrated that PLD is involved in the signaling cascade of cytokinins in plant cells [12,13].An application of BAP together with 1-butanol in amaranthus tissues induced the phosphatidylbutanol synthesis, catalyzed by PLD (Fig. 1).Further studies using transgenic plants will allow revealing PLD isoenzyme, playing a significant role in the signaling cascade of cytokinins.Abscisic acid (ABA) is a phytohormone, me di at ing the re ac tion of plant me tab o lism to wa ter de fi ciency [16].PLD plays a key role in the in duc tion of stomata clo sure, con di tioned by ABA.Upon knock out and knock down of PLDa1, stomata of Arabidopsis do not close un der the in flu ence of this phytohormone, while the ap pli ca tion of PA pro vokes their clo sure [16].Ar tifi cial in crease in PLDa1 gene ex pres sion re sults in increas ing stomata sen si tiv ity to ABA [16].Arabidopsis plants, which are PLDa1 gene knock out, do not demon strate any in crease in the lev els of PA and phosphatidylbutanol upon hy dro ly sis of phosphatidylcholine, stim u lated by ABA [16].The anal y sis of PA, formed un der the in flu ence of ABA at PLDa1 knock out, tes ti fies to the fact that PLDa1 deter mines rapid in crease in the con tent lev els of var i ous types of phos pha tid ic ac ids in the cells of leaves in response to this phytohormone [17].Pro tein phosphatase ABI1 is a neg a tive reg u la tor of ABA sig nal transduction, block ing the ex pres sion of genes in the nu cleus of cells, which are sen si tive to this phytohormone.ABA stim u lates the bind ing of ABI1 to plas matic mem brane and in hi bi tion of ac tiv ity of this en zyme, en sured by PA.In Arabidopsis plants, which are PLDa1 gene knock out, PA does not bind to pro tein phosphatase ABI1 and translocation of ABI1 to plasmatic mem branes does not oc cur which re sults in ac cumu la tion of this pro tein in nu clei of cells [16].ABA stim u lates PLDa1, thus ac ti vat ing the biosynthesis of PA, which bind and in hibit ABI1, that, in its turn, induces the ex pres sion of genes of re sponse to ABA.It was re vealed that mu tant plda1 is not sen si tive to ABA in the pro cess of ac ti va tion of stomata clo sure and in hibi tion of their open ing.How ever, in dou ble knock out mu tant (of genes PLD and ABI1) plda1abi1 ABA does not in hibit the open ing of guard cell ap er ture, but stimu lates its clo sure.This may be ex plained by the removal of in hib it ing ef fect of ABI1 on clo sure of stomata, stim u lated by ABA.Tak ing the abovementioned into con sid er ation, it can be stated that at knock out of ABI1 and PLDa1 genes the signaling cascade of stomata closure is inactivated, but the mechanism of suppression of their opening is inhibited [16,18].
PLDa1 binds to á-sub unit of het erotrimeric G-protein (GPA1) [19,20].The trans gen ic Arabidopsis plants with mu ta tions in plda1 and gpa1 genes are char ac ter ized by in creased loss of wa ter in leaves.Consid er ably weak ened sen si tiv ity to ABA in flu ence in the pro cesses of in duc tion of clo sure and block ing open ing of guard cell ap er tures was reg is tered in dou ble mu tant plda1gpa1 sim i larly to phe no type of plants plda1 [18].The plants ex press ing PLD, which is not ca pa ble of bind ing to a-sub unit of heterotrimeric G-pro tein (PLDa1 K564A ), are more sus cep ti ble to the in hi bi tion of open ing of guard cell ap er ture, stim u lated by ABA, com pared to the wild type plants.How ever, PLDa1 K564A plants are char ac ter ized by nor mal sen si tivity to ABA in the pro cess of stim u la tion of stomata closure.This tes ti fies to the fact that weak en ing of PLDa1 in ter ac tion with GPA1, re sult ing in their ac ti va tion [20], causes the in crease in re sponse to abscisic acid in the pro cess of in hi bi tion of stomata open ing, not af fecting the clo sure of guard cell ap er ture, in duced by ABA.The plants with knock out of gene of a-sub unit of heterotrimeric G-pro tein (gpa1) and dou ble mu tants plda1gpa1 dem on strate only stim u la tion of stomata clo sure upon the ap pli ca tion of PA.This phospholipid blocks stomata open ing in plda1-plants con trary to mu tants gpa1 and plda1gpa1 [18].
There fore, in the ab sence of a-sub unit of hetero-three-di men sional G-pro tein, PLD is not ca pa ble of im ple ment ing ABA ac tion on the block ade of stomata clo sure.Thus, PLDa1 and PA in mech a nisms of reg ula tion of stomata dy nam ics func tion prior to GPA1 and ABI1.The ac ti va tion of PLD in re sponse to the ABA ac tion re sults in the for ma tion of sec ond ary mes sen gers of sig nal ing cas cades.Knock out of PLDa1 gene prevents from the ac cu mu la tion of ac tive ox y gen forms (hy dro gen per ox ide) and ni tro gen ox ide, caused by ABA in stomata cells.The ac tiv ity of NADPH-oxidase, cat a lyz ing the for ma tion of hy drogen per ox ide in re sponse to ABA, is in hib ited in these trans gen ic plants.Hy dro gen per ox ide and do nor of nitro gen ox ide, in tro duced exogeneously, stim u late stomata clo sure in both trans gen ic and wild type plants [17].These re sults tes tify to the fact that ABA along with ac ti va tion of PLDa1 causes the for ma tion of phos pha tid ic ac ids.In their turn, they me di ate the ac tiva tion of sig nal ing cas cade of heterotrimeric G-proteins, which blocks stomata open ing, and in hibit protein phosphatase ABI1 and stimulate accumulation of hydrogen peroxide and nitrogen oxide, causing stomata closure.
The expression of genetic construction, containing anti-sense sequence of PLDb1 gene of rice, neutralizes seed dormancy, caused by ABA.An analysis of expression of the response genes to ABA in these transgenic plants revealed that PLDb1 is a stimulator of ABA signal transmission.This becomes evident through the activation of SAPK8 and SAPK10 expression as well as the decrease in the levels of transcripts of inducers of GAmyb and a-amylase germination.This mechanism is assumed to promote the inhibition of seed germination [4].Gibberellins stimulate the seeds to leave quiescence, in other words, they act in this process as ABA antagonists.Rice plants with PLDb1 deficiency are characterized by increased sensitivity to gibberellins [4].ABA inhibits cell division [21] and activates organ senescence [14].In Arabidopsis mutants, which are PLDa1 and PLDa4 genes knockout, contrary to the mutants of PLDb1 and PLDd1, the negative action of ABA on the expression ofKu gene is neutralized.The product of this gene plays a positive role in processes of DNA restoration and replication [22].The expression of anti-sense construction of PLDa1 suppresses senescence of leaves, induced by ABA [23].
Phospholipase D mutants in the analysis of mechanisms of ethylene and auxin action on metabolism of plant cells.Ethylene plays a key role in the plants response to stresses [24] and in the induction of fruit ripening [25].Suppression of senescence was registered in the leaves of Arabidopsis plants, where antisense construction of PLDa1 gene is expressed [23].These mutants have PLDâ and PLDã activity of wild type plants, which proves that the absence of functioning PLDa gene is not compensated by the presence of PLDâ and PLDã in cells.Therefore, PLDá1 is a determining factor in the realization of ethylene action on senescence of leaves.On the other hand, the expression of antisense construction of PLDa1 gene in tomatoes causes the delay in ethylene evolution and suppression of the process of fruit ripening [26].
Auxins reg u late the de vel op ment of ves sels, gravitropism of roots, as well as di vi sion and growth of cells [27].PLDz2-de fi cient plants are less sen si tive to auxin; they are char ac ter ized by in hib ited gravitropism of roots and auxin-de pend ent growth of hy po cot yls, while an op po site phe no type was reg is tered upon overexpression of the gene of men tioned isoenzyme which tes ti fies to the pos i tive role of PLDz2 in re actions of plant me tab o lism on the ac tion of auxin.Both the level of ex pres sion of early re sponse genes to auxin and the ac tiv ity of sen si tive pro moter DR5-GUS decrease in knock out or PLDz2-de fi cient plants and increase at ar ti fi cial in ten si fi ca tion of PLDz2 ex pres sion.It proves a role of PLDz2 in the re al iza tion of the auxin ac tion in plant cells [28].The trans port of intracellular ves i cles, filled with auxin, is blocked in PLDz2-de ficient plants, but it is stim u lated at ar ti fi cial in crease in the ex pres sion of PLDz2 gene [28].Pro tein PIN2 partic i pates in po lar trans port of auxin as a phytohormone trans porter and is sig nif i cant for reg u la tion of gravitropism of roots [29].Cy clic stream of PIN2-contain ing ves i cles in cells is blocked in PLDz2-de fi cient plants, but in ten si fied in roots with ar ti fi cially enhanced ex pres sion of the gene of this isoenzyme.However, ge netic ma nip u la tions with PLDz2 do not af fect po lar lo cal iza tion of PIN2 [28].As PLDz2 gene knockout causes only par tial weak en ing of cell re sponse to auxin (con trary to no ta ble sen si tiv ity loss in the mutants of transduction of this phytohormone sig nal), PLDz2 may in flu ence the auxin sig nal ing in di rectly due to reg u la tion of its trans port in plants [28].In apexes of roots PLDz2 plays a key role as a mod u la tor of trans port of this phytohormone.Auxin translocation is in hib ited by 40% in PLDz2 mu tants, but in creases by 30% in case of ar ti fi cial en hanc ing in the level of expres sion of PLDz2 gene [30].
Genetic manipulations with phospholipase D in research on the mechanisms of impact of oxidative stress and tissues wounding.Accumulation of active forms of oxygen in cells takes place in response to the action of various biotic and abiotic stresses [31].The expression of antisense construction of PLDa1 gene of Arabidopsis blocks the formation of superoxide radicals, catalyzed by NADPH-oxidase, while PA, applied in exogenous way, stimulate this reaction in the leaves of the mentioned plants [32].On the other hand, the protoplasts and leaves of plants, which are PLDd gene knockout, are characterized by increased sensitivity to hydrogen peroxide.An increase in the level of PA and phosphatidylbutanol as well as the activity of 49 kDa MAP-kinases under the influence of hydrogen peroxide are inhibited in plants, which are PLDd knockout.Moreover, the viability of plants at ultraviolet irradiation, stimulating the formation of hydrogen peroxide, decreases sharply at PLDd gene knockout [33].
Therefore, isoenzymes of PLD participate in response to reactive oxygen forms: PLDa1 stimulates their formation, while PLDä provides metabolism reaction in response to their action.
In Arabidopsis with the expression of antisense construction of PLDa1 gene, the cell wounding as a stress factor causes inhibition of the formation of PA and jasmonic acid, as well as the expression of gene of LOX2 lipoxigenase [34].
Moreover, local accumulation of PA and increase in the PLD activity in the leaves of plants, destroyed due to wounding, are completely eliminated in double knockout mutants PLDa1/PLDd.Knockout of PLDa1 and PLDd genes testifies to a significant role of these isoenzymes in PA accumulation, conditioned by the wounding.However, the expression of LOX2 gene, levels of jasmonic acid and its predecessors, as well as the activity of MAP-kinases do not change in double mutants PLDa1/PLDd [35], which does not confirm the significance of these isoenzymes for abovementioned defence reactions of metabolism of plant cells to the wounding.
Isoforms of phospholipase D, participating in the realization of signaling at biotic stresses.Sensitivity and resistance of plants to specific pathogens depend on the plants recognition abilities [36].The role of PLD in the mechanisms of plant interaction with pathogens was studied in tomatoes with PLDb1 gene knockdown.It was revealed that in response to the action of xylanase elicitor the mentioned isoenzyme is a negative regulator of the formation of reactive oxygen species, but a positive regulator of the expression of gene of b-D-xylosidase, which is an enzyme, participating in restructuring of plant cellular wall [37].However, in the absence of infection the knockdown of PLDb1 gene of rice determines activation of defence reactions of plants, i.e. accumulation of reactive oxygen species and induction of expression of defensive response genes (PR-1, PR-4, â-glucanases, chitinases, traumatin-like protein, transcription factors of WRKY and ERF families).Death of the cells, specific for hypersensitivity reaction, as well as biosynthesis of phytoalexins and increase in resistance to pathogens -Pyricularia grisea fungi and Xathonomonas oryzae bacteria -were registered in these transgenic plants [38].These data testify to the role of PLDb1 as a negative regulator of defensive reactions and resistance of plants at under normal growth conditions and as a positive one-at pathogens action.
The analysis of metabolism reaction of cells to cold stress on the example of Arabidopsis plants, transgenic in phospholipase D genes.Twice-weakened formation of PA, caused by freezing, was revealed in the plants, expressing antisense construction of PLDa1 gene, which is also specific for phosphatidylcholine breakdown.Increase in the resistance to freezing was registered for PLDa1-deficient plants [39].
On the contrary, isoenzyme PLDd, bound to plasmatic membranes, promotes resistance increase of Arabidopsis to freezing.Gene knockout of this PLD isoenzyme causes sharp decrease in plant resistance to cold, while artificial enhancement of the expression of PLDd gene increases freezing tolerance of plants [40].Contrary to PLDa1, PLDd does not promote mass decomposition of membrane lipids, but causes formation of PA, performing a signaling function in the adaptation of cell metabolism to low temperatures.Plants, which are PLDd gene knockout, grow and develop at under normal conditions without evident changes [40,41].On the other hand, these transgenic plants are more sensitive to the oxidative stress [33].
PLDa1 plays a key role in the process of hydrolysis of phosphatidylglycerol of plastids, while PLDä functions as a negative regulator of decomposition of plastid lipids and PA accumulation under these conditions [42].PLD are the modulators of expression of genes of plant tolerance to cold.Artificial expression of antisense construction of PLDa1 results in the increase in resistance to freezing in both acclimatized and non-acclimatized Arabidopsis plants.The activation of expression of physiological response genes to low temperatures -COR47 and COR78 -was not revealed in non-acclimatized PLDa1-deficient plants.However, intense expression of COR47 and COR78 genes as well as increase in the osmolytes level were revealed in PLDa1-deficient plants at conditions of acclimatization to low temperatures and especially to freezing [43].Still, at low temperatures PLDd knockout does not change the expression of COR genes, providing resistance to freezing.PLDd also forms PA, weakening cell damage, caused by oxidative stress at low temperatures.
The role of PLD in the mechanisms of regulation of plant cell metabolism in response to the action of heat shock has not been fully specified yet.The increase in environment temperature to 40°C results in rapid increase in PA levels in cultures of tobacco cells, Arabidopsis sprouts and rice leaves.It is partially mediated by the activation of phospholipase D -the amount of phosphatidylbutanol is sharply increased at early stages of heat shock action [44].The abovementioned testifies to the significance of PLD for the transduction of the stress signal in plant cells.However, PLD isoenzymes, activated under the influence of heat shock, are still unknown.
Lipid sig nal ing at os motic stress.PLDa1 gene knock out in Arabidopsis in creases sen si tiv ity of plants to drought.Changes in ex pres sion of var i ous genes, cod ing en zymes of me tab o lism and transduction of phytohormone sig nals were re vealed at stress con ditions [45].Gene knock out or ex pres sion of antisense PLDa1 con struc tion in Arabidopsis re sults in in hi bition of sen si tiv ity of plants to abscisic acid, weak en ing of stomata clo sure, and in crease in wa ter loss af ter transpi ra tion [15,16,23].At the same time, ar ti fi cially inten si fied ex pres sion of PLDa1 gene in creases sus cep tibil ity of plants to ABA ac tion [15,16].PLDa1 play a pos i tive role in the in duc tion of clo sure of guard cell ap er ture in re sponse to ABA and in the de crease in water loss of plants after transpiration [16,18].
According to the results of phenotype analysis of knockout mutants, PLDa1 is a modulator of various reactions of plants to drought.This isoenzyme plays the role of stimulator of stomata closure at early stages of the mentioned stress [46].On the other hand, knockout or overexpression of PLDa3 gene does not influence the transpiration level [47] which testifies against the possibility of participation of this PLD isoenzyme in the regulation of dynamics of guard cell aperture in response to the action of abscisic acid.Moreover, the biosynthesis of this phytohormone does not change in PLDa3 knockout plants [47].ABA plays the role of a positive regulator of hydrotropism of plant roots.PLDx2 gene knockout violates and prevents hydrotropism of roots in Arabidopsis.Drought and abscisic acid, the accumulation of which is conditioned by this stress, stimulate the activity of PLDx2 promoter in root cap and block gravitropism of roots [48].Contrary to PLDa1 gene mutants, the level of accumulation of PA and phosphatidylbutanol in transgenic Arabidopsis plants with antisense PLDd construction decreases at dehydration conditions, which demonstrates the significance of PLDd for the formation of PA at the mentioned stress.However, notable changes in phenotype at both normal conditions and dehydration were registered neither in wild type plants nor in plants with PLD gene in antisense orientation [41].On the other hand, PLDd gene knockout in Arabidopsis increases the sensitivity of plants to salt stress [49].Therefore, PLDá1 and PLDä play their own roles in the process of cellular transmission of information about the conditions of water potential in the environment.Combined knockout of PLDa1 and PLDd genes causes the decrease in resistance of root growth to salt and hyperosmotic stresses.The double mutants of Arabidopsis PLDa1/PLDd are characterized by more evident sensitivity to salt stress compared to the gene mutants of each of these PLD isoenzymes.Knockout of PLDa1 or PLDd gene causes partial decrease in the level of PA, stimulated by salt stress, while at simultaneous knockout of both genes PA accumulation is only one-third compared to wild type plants [49].The final formation of phosphatidylbutanol and PA was revealed at conditions of salt stress and dehydration in the double mutants [49] which testifies to the participation of other PLD isoenzymes (presumably PLDa3 [47]) in the realization of the action of these stresses on plants.Moreover, PLDâ1 isoenzyme may also participate in reactions of plant cells to osmotic stress as rice with deficiency of the gene of this enzyme is characterized by increased sensitivity to high concentrations of salts [4].
PLDa3 plays an absolutely different role in the regulation of growth of plants at conditions of hyperosmotic stress.PLDa3 knockout Arabidopsis is characterized by intense expression of response genes to abscisic acid and increased sensitivity to salt stress and dehydration, while artificial increase in the expression level of gene of the mentioned isoenzyme increases plant resistance to these stresses.At this isoenzyme gene knockout the growth of roots is more inhibited under the influence of ABA contrary to the wild type plants.Artificially intensified expression of PLDa3 at the conditions of salt stress results in the increase in the number of side roots, acceleration of seed germination, growth of sprouts and roots.The growth of these transgenic plants is more expressed compared to the wild type at long-term salt stress [47].Artificial changes in PLDa3 expression at the conditions of salt stress and water deficiency are respectively reflected at the levels of PA and gene expression (TOR and AGC2), the products of which are modulators of growth processes in response to the incoming nutrients and action of stresses.The state of phosphorilation of protein of ribosomal protein kinase S6K changes as well [47].Therefore, PLDa1 and PLDa3 participate in the modulation of response of plants to the action of osmotic stress due to various mechanisms: PLDa1 mediates the action of abscisic acid on dynamics of guard cell aperture to decrease the level of water loss by plants, while PLDá3 causes the growth of roots in response to the action of salt stress and drought to enlarge the surface of water absorption.
The analysis of phenotype of plants at knockout and artificially intensified expression of PLDe gene testifies to the fact that PLDe stimulates the growth of Arabidopsis roots at the conditions of hyperosmotic stress, induced by water deficiency and high concentrations of salts.At the same time PLDe may have a key role in the mechanisms of plant perception of the state of provision with nutrients, mineral nitrogen, first and foremost [50].
Participation of PLD in the regulation of various stages of plant ontogenesis.PA, the product of PLD activity, is the central mediator of the biosynthesis of phospholipids [51].High temperature and humidity as well as long-term storing accelerate ripening of seeds.However, knockout or knockdown of PLDa1 gene in Arabidopsis stimulates their germination, blocks the decrease in the number of unsaturated fatty acids and decreases the level of accumulation of peroxide forms of lipids, thus improving quality of seeds.Sprouts of these transgenic plants grow faster and have longer roots and larger leaves.PLDa1 knockdown also causes plant seeds to lose their capability of ripening compared to plants, which are knockout of this isoenzyme gene, observed as the increase in their viability [52,53].On the other hand, artificial violation of the formation of functional protein PLDd promotes ripening of seeds and increases their germination capability [52].
The role of PLD in the regulation of reproductive process in plants has not been studied well enough.Artificially intensified expression of PLDa1 gene in Arabidopsis does not influence the formation of flowers.No differences in the number of seeds and pods compared to wild type were found in plants with the expression of antisense construction of this gene [23].The flowers of plants, expressing antisense construction of PLDa1 gene, contain only 10% of activity of the wild type PLD which evidences to self-sufficiency of the activity of other PLD isoenzymes to support normal development of abovementioned reproductive organs.These plants are characterized by the activity of PLDb and PLDg.However, the absence of reliable changes in seed formation [23] testifies to non-participation of PLDá1 in reproduction of plants.On the other hand, according to the researches on phenotype of knockout plants, PLDá1 participates in the formation of a basic level of PA in flowers, pedicles, seeds, but not pods of Arabidopsis [53].Contrary to the wild type plants, the plants with overexpressed PLDa3 are notable for early formation of flowers, seeds, and increased number of pods only at conditions of moderate drought.An absolutely opposite phenomenon was revealed at PLDa3 gene knockout.The expression of genes, coding the factors of induction of flower formation (TSF, BFT), is enhanced at artificial increase in the level of PLDa3 expression at the conditions of water deficiency, but decreases in plants, which are PLDa3 knockout.At normal conditions the expression of TSF gene only decreases at PLDa3 knockout compared to wild type plants [47].
It was shown that PLDae1 participates in the regulation of morphogenesis of root hair.Artificial intensification of PLDx1 gene expression causes the formation and enlargement of branching of ectopic root hair.The expression of antisense PLDx1 construction results in violation of localization, growth, and development of root hair, not influencing their laying.PLDx1 is assumed to regulate the growth and development of roots, modulating the processes of membrane transport, in particular, exocytosis [54].Different data were obtained at PLDx genes knockout.Localization of root hair has no evident changes in plants, which are PLDx1 and PLDx2 genes knockout, while extremely weak changes in the mentioned phenotype were revealed on apexes of roots at the conditions of low content of mineral phosphorus [55].One of the probable reasons of differences in the results of these researches may be experiment conditions: the system of induced repression of PLDx1 gene was used in the first case [54], and a knockout -in the second [55].It is possible that blockade of PLDx1 with the expression of antisense construction of the respective gene is not specific for PLDx1-other PLD isoenzymes may also be repressed in this case.
The second reason of differences in the results is the fact that a definite threshold of PLDx1 protein levels is required for laying and growth of root hair, while the expression of antisense construction of this gene may neutralize these parameters in cells only partially.However, PLDx may be significant for the regulation of growth of roots in response to stressful conditions of environment.The growth of primary roots at conditions of low concentrations of mineral phosphorus in the incubation medium in Arabidopsis plants, which are simultaneously knockout of PLDx1 and PLDx2, is inhibited compared to the wild type plants or the plants, knockout of one of PLDx genes.The growth of lateral roots increases in the double mutants and localization of the formation of root hair does not change.PLDx2 knockout decreases the level of PA accumulation in the roots at the conditions of mineral phosphorus deficiency [55].The morphology of root meristem at low content of phosphorus is sharply violated in the plants, which are PLDx2 gene knockout, that reflects on the growth of primary roots and root hair [56].
On the other hand, the growth of roots at knockout of PLDa1 and PLDa genes of Arabidopsis does not change significantly [49], PLDa1 may participate in the formation of the basic level of PA in Arabidopsis roots [53].Therefore, PLDx1 and PLDx2 simultaneously play the key role in the regulation of growth of roots in response to conditions of mineral phosphorus deficiency.
PLDe is a stimulator of the growth of lateral roots and accumulation of biomass in plants [50].At artificially intensified expression of PLDe gene, the lateral roots grow faster compared to the wild type plants, while PLDe knockout suppresses growth of roots.The number and growth of cells increase at artificially intensified expression of PLDe gene.The surface area of lamina, growth of leaf cells, number of leaves and their cells, as well as the number of seeds increase in these transgenic plants, but decrease at PLDe gene knockout.Plant sprouts grow faster at overexpression of PLDe gene, but grow slower in knockout plants compared to the wild type plants.The levels of PA content increase in leaves and roots upon overexpression of the gene of this isoenzyme, decreasing in the knockout plants [50].
The violation of membrane integrity is one of the main reasons of induction of senescence processes [57].Contrary to the wild type plants, the leaves of plants, where antisense construction of PLDa1 gene is expressed, are characterized by weakening in senescence features, conditioned by abscisic acid and ethylene [23].ABA and ethylene play a significant role in reactions of plants to the action of stresses increasing biosynthesis of these phytohormones [58,59].Therefore, catabolism of membrane lipids, performed by PLDa1, may be the result of response to stresses, and not the reason of natural senescence.
The role of PLD in the processes of growth and ripening of fruit may be revealed in the regulation of metabolism of their cells [60].Tomato fruits, where antisense construction of PLDa1 gene is expressed, are smaller in size compared to those of the wild type plants, they are kept longer, are harder and notable for delayed climacteric evolution of ethylene [26].
Therefore, various isoenzymes of PLD participate in the regulation of growth and development of plants.
Conclusions.The results of recent researches using transgenic plants have significantly extended the ideas on the mechanisms of realization PLD-induced signals in the cell (Fig. 2).A wide spectrum of PLD isoenzymes in plants provides the formation of various types of phosphatidic acids in specific organs and cells in the process of their development at the action of external stimuli.This ensures the variety of PA functions in plants, since it is a secondary messenger of signaling cascades, modulating the activity of key enzymes in response to the action of stresses (osmotic, cold, oxidative) and phytohormones (ABA, auxins, cytokinins, and ethylene).At the same time little progress has been achieved in the understanding of the interaction of PLS and PLD and the role of diacylglycerolkinases in PA synthesis.It is also important to analyze the nature of PA interaction with the targets of signaling systems, the recognition of which is possible with the elaboration of a biosensor for PA determination, combining a specific factor of PA binding with fluorescent protein.The application of the biosensor would allow obtaining the information about components of signaling systems of lipid nature and their role in the coordination of intensity and direction of cell metabolism, as well as in the realization of genetic programmes of plant growth and development.
The work was supported by the grants of NAS of Ukraine