The molecular components of phospho-and glycolið 3 d metabolism in p lant cell membranes under the phosphorus deficiency

One of the aspects of molecular regulation of phosphorus metabolism in plants, the lip3d components of membrane structures, has been reviewed. The refocusing of phosphoand glycolip3d metabolism is an indicator of phosphorus accessibility in plants. The compensatory mechanisms of substitution of phospholip3ds with non-phosphorus containing glycolip3ds in membranes, allow plants to adapt to the phosphate (Ð3) starvation. Phospholip3ds are the reserve pool of cellular phosphorus at reutilization of ions in the donor-acceptor system of plants. The mechanisms of transcriptional regulation of genes involved in the synthesis of phospholip3ds and glycolip3ds under Ð3 deficit have been analyzed.

The maintenance of homeostasis and the metabolism control are defined by the cellular signalling in the donor-acceptor system of plants.While a plant organism responds to the change in the environmental conditions, the key role in the control of cell signalling is attributed to phosphorus.Being an essential structural and functional component of many key macromolecules, nucleic acids, highly energetic structures (AMP, ADP, ATP), membrane phospholipids, phosphorus participates in many metabolic processes in plants: energy transfer, carbohydrate assimilation, breathing, lipid biosynthesis and regulation of enzyme activity [1][2][3][4].
In the process of evolution the plants developed different adaptation mechanisms in response to phosphorus (P i ) deficiency.The decrease in the concentration of phosphorus ions in the plant cell to the critical level is a signal to launch reciprocal stress, causing an activation of the alternative metabolic pathways, directed towards the mobilization and decreased use of phosphorus in plants [3,4].The maintenance of constant phosphorus concentrations in the organism is achieved via the input of P i from outside, storing, re-mobilization and re-utilization of phosphorus in the donor-acceptor system of plants in accordance to the priorities in the distribution of assimilates during their growth and development [4,5].
One of the regulatory mechanisms of maintaining optimal concentrations of phosphorus ions in the plant cell is related to the modification of membranes structure and the change in the rate and direction of metabolism of lipid components in all subcellular compartments.
SQDG in plant cells is localized only in plastid membranes while PG is present in plastid membranes as well as and others though in insignificant quantities.PG is the only phospholipid, contained in thylakoids and in the internal membrane of chloroplasts.A third of organic phosphates in plants, in Arabidopsis thaliana in particular, belong to phospholipids [9].
The developed system of membrane structures and the diversity of membrane lipid composition allow the cells to function with minimal phosphorus consumption which is crucial for plants since the deficiency of this chemical element is often restrictive for their growth and development [3,10].
The transcriptional control of metabolism of phospho-and glycolipids under P i deficiency.The genes, controlling synthesis, degradation and transformation of lipid components, phospho-and glycolipids, in particular, are considered as an essential constituent of the plant regulatory system, involved in the response to P i deficiency.The quantitative changes in the phospho-and glycolipids are an important index of P i deficiency in plants (Table ).
MGDG-synthase, type A, is expressed in photosynthetic tissues during the plant growth and development in the presence of P i .It is responsible for the mass synthesis of MGDG, required for the biogenesis of internal membranes of chloroplasts and expansion of the network of thylakoids.
MGDG synthase, type B, does not participate in the galactolipid synthesis under conditions of P i saturation and is expressed only at its deficiency in plants.
MGDG-synthase, synthesized with the participation of MGD gene type B, is mainly localized in non-photosynthetic tissues: in inflorescences (MGD2) and roots (MGD3).The expression of MGD gene of this type is essential for the launch of alternative pathway of biosynthesis of galactolipids at P i deficiency.
The functional distribution between the internal (MGD1) and external membranes (MGD2/3) of chloroplasts, corresponding to the degree of phosphorus provision for plants, is mainly controlled by the level of phytohormones and light intensity.In particular, the expression of MGD1 gene is regulated by light intensity and content of cytokinins.The P i -dependent expression of MGD2/3 is inhibited by cytokinins and induced by auxin-dependent signalling pathways [15,16].
The DGDG biosynthesis is regulated by the transcription of genes of DGDG-synthases (EC 2.4.1.241):DGD1 and DGD2 [17].The study of plants dgd1/pho1 demonstrated that the mutation PHO1, blocking the input of P i into the xylem, in combination with the mutation DGD1 leads to the restoration of DGDG biosynthesis [18].Since DGD1 mutation is characterized by the presence of stop-codon in the site of coding DGD1, which results in the 90 % decrease in DGDG biosynthesis [12], the restoration of its content testifies to the presence of DGD1-independent pathway of galactolipid biosynthesis.The creation of genetically modified A. thaliana plants dgd1 and dgd1/pho1 resulted in the identification of the second gene, responsible for the DGDG synthesis ?DGD2, activated at P i deficiency [17].
The construction of a model system in vitro using MGDG and uridine-5-diphosphate (UDP)-galactose as a substrate promoted the study on DGDG synthase activity.It was determined that UDP-galactose (EC 2.4.1.46)and not MGDG is a galactose donor for DGDG, the synthesis of which is conditioned by the level of DGD2 expression.The creation of this model system confirmed the dependence of DGDG synthesis on the availability of UDP-galactose in higher plants [19].
The presence of an additional pathway of galactolipid synthesis, independent from the transcription of DGD1 and DGD2 genes, was discovered in modified plants dgd1/dgd2.Since plants Reduction of MGDG synthesis in leaves to 75%; presence of yellow and green leaves due to disorder of biogenesis of chloroplasts [11,15,39,50] MGD2 (At5g20410) Biosynthesis of MGDG in P i absence; no participation in MGDG synthesis in P i presence; localization on the external membrane of chloroplasts; encoding MGDG synthase, type B (UDPgalactoso-1,2-DAG-galactosyl transferase); induction in non-photosynthetic tissues Reduction of DGDG content and change in the composition of fatty acids of galactolipids in leaves and roots at P i deficiency; no biochemical and phenotypic changes are revealed at optimal supply of P i [11,15,39,50] MGD3 (At2g11810) Biosynthesis of MGDG in P i absence; no promotion of synthesis of galactolipids in P i presence; localization on the external membrane of chloroplasts; participation in metabolic processes of fatty acids; encoding MGDG-synthase, type B (UDP-galactose-1,2-DAG-galactosyl transferase) Reduction of DGDG content and change in the composition of fatty acids of galactolipids in leaves and roots at P i deficiency; no biochemical and phenotypic changes at optimal supply of P i [11,15,39,50] Biosynthesis of DGDG; localization on the external membrane of chloroplasts, in mitochondria; provision of the ultimate composition of galactolipids in photosynthetic membranes; stabilization of subunits PsaD, PsaE of the main PSI complex; encoding synthase DGDG (UDP-galactoso-MGDG-galactosyl transferase); catalysis of galactose transfer from UDP galactose to the acceptor molecule Reduction of DGDG synthesis to 90%; diminution of growth, defects in seed color, pale leaves, reduction of photosynthetic potential and change in thylakoid structure (formation of "convoluted" thylakoids) [12,39,51] Biosynthesis of DGDG; localization on the external membrane of chloroplasts; encoding DGDG synthase (UDP-galactoso-MGDG-galactosyl transferase); catalysis of galactose transfer from UDP-galactose to the acceptor molecule Absence of phenotypic specificity under normal conditions of cultivation [12,17,39] Degradation of phospholipids and synthesis of galactolipids in roots; encoding subfamily of proteins PXPH-PLD of phospholipase D; regulation of root architecture at P i deficiency Inhibition of growth of the main root and elongation of side roots at P i deficiency [34][35][36] PLDz2 (At3g05630) Production of phosphatidic acid at P i deficiency; induction in roots and rosettes at P i deficiency; encoding subfamily of PXPH-PLD proteins of phospholipase D; promotion of hydrolysis of PC and PE with the formation of DAG; no regulation of root fibril architecture at P i deficiency Disorder of hydrolysis of phospholipids, reduced capability of accumulating galactolipids; changes in root morphology at P i deficiency [34][35][36] Transcriptional regulation of A. thaliana genes, participating in the synthesis and transformations of glyco-and phospholipids at P i deficiency dgd1 and dgd2 carry null mutations, they have an alternative third way of DGDG synthesis, related to galactolipid galactosyltransferase (EC 2.4.1.184),localized in chloroplast membranes and synthesizing DGDG from MGDG in the absence of UDP-galactose.This alternative way is not related to the synthesis of galactolipids in plants in optimal nutrition conditions [17].
Synthases MGDG (type B) and DGDG, synthesized as a result of expression of genes MGD2, MGD3 and DGD2 in conditions of P i deficiency, are in the external membrane of chloroplasts [13,17].All the predecessors, participating in MGDG synthesis, are transported from the endoplasmatic reticulum (ER) due to the absence of the active way of galactolipid synthesis in plants, notable for prokaryotes [18].
It is noteworthy that DGDG, synthesized in conditions of P i deficiency, is mainly localized in extraplastidic membranes; the enzymes, involved in the biosynthesis of this galactolipid, are located in plastid and other membranes.Therefore, the genes MGD2, MGD3 and DGD2 are likely to be involved in the biosynthesis of galactolipids of extraplastidic membranes, ER in particular, though at present there is no direct evidence confirming this fact.
The genes of PGP family control the biosynthesis of phospholipids.The isoenzymes of phosphate-synthase PG (EC 2.7.8.5) in A. thaliana plants are encoded by two genes: PGP1 and PGP2 [20].PGP1 encodes the predecessor of the enzyme, localized in both plastids and mitochondria.The synthesis of microsome isoenzymes is controlled by PGP2 [20,21].The significance of this gene for the biosynthesis of PG in plastids is demonstrated on modified plants of A. thaliana, where PGP1 is partially or completely inactivated [21][22][23].Contrary to plastids, PG deficiency in mitochondria is likely to be compensated by its delivery from ER where PG is synthesized with the participation of PGP2 gene [21].
DAG, synthesized in plastids, is the substrate for the phosphate synthase of PG of photosynthetic membranes.At the same time, similarly to the synthases MGDG and DGDG, the synthase SQDG can use as a substrate DAG, synthesized not only in plastids, but also imported from ER [30].This capability of the glycolipid synthases, together with the expression of specific genes, required for the DAG transformation due to phospholipid degradation into SQDG and DGDG, is the molecular foundation for the significant transformations of the membrane lipid composition at P i deficiency [11,17,24,25,29,[31][32][33].Èçìåíåíèå ñòðóêòóðû õëîðîïëàñòîâ, íî ïðè ýòîì íàáëþäàþòñÿ íîðìàëüíûå ìèòîõîíäðèè; áëåäíî-çåëåíàÿ îêðàñêà [20,21]

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The genes, controlling the synthesis of phospholipases, are involved into the degradation of phospholipids, the synthesis of galactolipids, DAG and phosphatidic acid (PA) in plants.In particular, PLDz genes encode the subfamily of PXPH-PLD proteins of phospholipase D (EC 3.1.4.4), which causes the phospholipid degradation and galactolipid formation in both photosynthetic and non-photosynthetic cells.The activation of PLDz1 transcription results in the degradation of phospholipids in roots.PLDz2 , expressed at P i deficiency in both roots and rosettes, initiates PC and PE hydrolysis with the formation of DAG [34][35][36].
The biosynthesis of non-specific phospholipase C (EC 3.1.4.3) is controlled by the transcription of NPC5 gene.In case of P i deficiency the phospholipase C causes the degradation of phospholipids and the synthesis of galactolipids synthesis in plant leaves [37].
The study on phosphatase and nuclease activity of genetically modified A. thaliana plants, cultivated at different P i concentrations, and subsequent identification of the genes present valuable information about the molecular mechanisms of regulation of P i homeostasis.The application of differential or subtractive hybridization of DNA-microchips revealed that the transcriptional level of several hundreds of genes is regulated by the change in P i concentration [38].The functions of these genes are rather different which highlights the relevance of phosphorus for optimal functioning of cells.The results of various manipulations with genes allowed deepening the knowledge about their regulatory role in P i homeostasis.
An analysis of the level of gene transcription showed that at phosphorus deficiency 44 genes are involved in the biosynthesis of lipids of A. thaliana plants (7%).Only two of them turned out to be suppressed.Approximately 50% genes, related to the lipid exchange, are expressed during two days after the beginning of P i deficiency.Primarily these are genes, encoding the enzymes, involved into the degradation of phospholipids and synthesis of galacto-and sulfolipids as well as into the DAG biosynthesis (Fig. 1) [39,40].The transcriptional regulation only a few of them, participating in the biosynthesis of phospholipases D (PLDz2) and C (NPC5), is induced in the conditions of P i deficiency [40,41].
The genes MGD2 and MGD3 are expressed 4-10 times faster, compared to DGD1 and DGD2, the induction of which, in its turn, is observed only after the action of medium-and long-term P i deficiency.Besides, the genes, encoding UDP-galactose i.e.UDP-glucose-4-epimerase and UDP-galactose-4-epimerase (UGE2 and UGE5), which transform UDP-glucose into UDP-galactose (the predecessors of galactolipids), are also expressed at medium-and long-term P i deficiency.For comparison: the genes, coding UDP-sulfoquinovose -UDP-sulfoquinovosyl synthase and UDP-sulfoquinovosyl-DAG-sulfoquinovosyl transferase (SQD1 and SQD2), are activated at both short-and long-term P i deficiency, which resulted in 4-fold increase in the SQDG level at long-term P i deficiency.
Such modulations in the regulation of transcription of the genes, participating in the biosynthesis of glycoand phospholipids, confirm the hypothesis of complex mechanism of substitution of membrane phospholipids with non-phosphorus-containing glycolipids in plants at P i deficiency [40].
The re-utilization of phosphorus of membrane phospholipids in conditions of P i deficiency.The substitutions of phospholipids with non-phosphorus-containing lipids in the membranes was first discovered in non-photosynthetic bacteria Pseudonomas diminuta [42] and then in photosynthetic organisms [43].In genetically modified photosynthetic bacteria (Rhodobacter sp. and Synechococcus sp.), where non-phosphorus-containing SQDG is absent, its accumulation was found at P i deficiency [43,44].The reversible interrelation between SQDG and PG, depending on the level of P i provision, was also determined in A. thaliana plants [25].The increase in SQDG content along with the PG destruction was established in Chlamydomonas reinharditti at P i deficiency [45].These observations became a foundation of the hypothesis of substitution of PG forSQDG in photosynthetic membranes [27,29].
The investigations of mutant plants A. thaliana pho1 deficient in a protein, providing the input of P i into the xylem [9], testify that at P i deficiency the content not only of SQDG, but also of DGDG increases.The accumulation of SQDG correlates with the increase in the protein content which proves an indirect participation of the latter in SQDG synthesis [18,46].
An analysis of subcellular fractions of plastidic and extraplastidic membranes of A. thaliana revealed considerable increase in DGDG content in the membrane fraction of roots in conditions of P i deficiency in both wild and transformed plants dgd1 [18].Along with an intense accumulation of DGDG in the fraction of non-photosynthetic membranes, less considerable increase in the relative content of this galactolipid was also observed in the membranes of chloroplasts of P i -deficient non-modified and dgd1-transformed plants.
For fad3-modified A. thaliana plants with inadequate synthesis of desaturase C 18:2 of the fatty acid (FA) associated with ER the accumulation of C 18:2 was found as well as a decrease in the level of C 18:3 of FA in the composition of DGDG of these plants.Since FAD3 mutation is primarily related to lipids, present in extraplastidic membranes, the changes in the ratio C 18:2 : C 18:3 of FA DGDG of fad3-plants testify that DGDG is mainly localized in extraplastidic membranes [47].
Therefore, the obtained results confirmed the predominant accumulation of DGDG in plasmatic membranes of plants, cultivated at P i deficiency, which still does not rule out the possibility of this galactolipid accumulation in photosynthetic membranes as well.
The main lipids in extraplastidic membranes are galactolipid DGDG and phospholipid PC.They are neutral compounds, involved in the formation of the membrane bilayer.The analysis of FA in the composition of these lipids revealed their similarity.Therefore, it was assumed that PC, synthesized at P i deficiency, in plasmatic membranes substitutes DGDG, like SQDG compensates PG in the membranes of chloroplasts [25,46].
The comparative analysis of transformation of lipids of plasmatic membranes and chloroplast membranes demonstrated that short-term P i deficiency does not cause any significant modifications in the composition of neutral lipids of plasmatic membranes.At insignificant changes in PC there was no intense accumulation of DGDG in the membranes of roots of A. thaliana plants during two-day P i deficiency [40].The data obtained show a lesser sensitivity of nonphotosynthetic membranes compared to chloroplast membranes at P i deficiency [48].
The re-utilization of phosphorus from phospholipids of membranes at P i deficiency is reversible.The studies on Avena sativa L. plants [demonstrated that at the renewed input of P i into plants of Pi deficiency according to [40]: -short-term Pi deficiency (up to 12 hours); -medium (up to 2 days); -long-term (over 2 days, leaves); -long-term (over 2 days, roots).Quantitative changes (times): black -> 10; dark grey -4-10; light grey -2-4; white -0.5-2; stripes < 0.25.HCP = 0.05 a radioactively-labeled phosphorus is included first of all in the PC molecules and two days later over a half of phospholipids of plasmatic membranes in roots contained labeled phosphorus [49].
Investigation of functional activity of phospholipases, using the analysis of amino acid sequence of bacterial phospholipase C, similar to that of A. thaliana, revealed six phospholipases Cs.Considerable activation of transcription of only one of them, non-specific phospholipase C4 (EC 3.1.4.3) (NPC4) [,] was observed at P i deficiency.Using molecular cloning and functional expression of NPC4, it was confirmed that this gene participates in encoding PC-hydrolysing phospholipase C4, the functional activity of which does not depend on the presence of Ca 2+ ions [31].The study of the activity of phospholipases C in A. sativa did not reveal their intense participation in the degradation of phospholipids of plasmatic membranes of A. sativa roots [49].
The presence of npc4 mutation in A. thaliana plants causes significant decrease in the PC-hydrolysing activity of phospholipase C due to P i deficiency.The data obtained allow an assumption about the participation of NPC4 in the delivery of both non-organic phosphate and DAG, formed due to degradation of phospholipids in plasmatic membranes at P i deficiency [31].
Besides the activation of transcription of phospholipase C4, the increase in the activity of non-specific phospholipase C5 (NPC5) was determined in A. thaliana plants at P i deficiency.The analysis of the lipid fraction in mutant plants of A. thaliana demonstrated that the functional activity of NCP5 gene has significant] impact on the biosynthesis of DGDG in photosynthetic membranes at P i deficiency.The biosynthesis of DGDG in transformed plants npc5/pho1 decreased considerably compared to non-modified variants.The data obtained allowed the authors to make a conclusion about the dependence of approximately 50% of DGDG synthesis in photosynthetic membranes on the functionality of NPC5 gene at P i deficiency [37].
The determined functional activity of phospholipase D is in close correlation to the ratio of DGDG/PC at different levels of P i provision for plants.
The established correlation between the activity of phospholipase D and DGDG/PC ratio is in good agreement with the results, obtained on the model of substituting phospholipids with DGDG with the formation of PC in plasmatic membranes [49].
The studies on the intensity of phospholipid hydrolysis in roots and rosettes of A. thaliana determined their more intense degradation in plasmatic membranes of roots.Research of the activity of phospholipase D in A. thaliana plants on the example of genetically transformed plants pldz1, pldz2 and pldz1/pldz2 demonstrated that the disorder of functions of PLDz1 and PLDz2 leads to the reduction in PC degradation along with the diminution of DGDG accumulation in P i -deficient plants.It was shown that PC hydrolysis with the participation of PLDz at P i deficiency promotes the delivery of non-organic phosphorus for cellular metabolism and DAG -for the synthesis of galactolipids [35].
Thus, the complex approach using genetic, biochemical and physiological methods to study the transformation of glyco-and phospholipids revealed compensatory mechanisms of substitution of phospholipids with non-phosphorus-containing glycolipids in plastid and plasmatic membranes, which are manifested as a capability of plants to react to P i deficiency by selective accumulation of SQDG and DGDG (Fig. 2).The modification of lipid components of membranes, directed towards the synthesis of non-phosphorus-containing glycolipids in response to P i deficiency, promotes the maintenance of developed systems of chloroplasts and plasmatic membranes of plants.Besides, phospholipids serve as a reserve pool of cellular phosphorus.The described re-utilization of phosphorus ions in the donor-acceptor system is one of the strategies allowing plants to adapt to the conditions of limited P i .