The Use of RNA Interference for the Metabolic ... - Springer Link

ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 2, pp. 146–156. © Pleiades Publishing, Ltd., 2010. Original Russian Text © E.B. Rukavtsova, V.V. Alekseeva, Ya.I. Buryanov, 2010, published in Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 2, pp. 159–169.

On 50year anniversary of the Institute of Bioorganic Chemistry

The Use of RNA Interference for the Metabolic Engineering of Plants (Review) E. B. Rukavtsova1, V. V. Alekseeva, and Ya. I. Buryanov Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Division, Russian Academy of Sciences, pr. Nauki 6, Pushchino, Moscow oblast, 142290 Russia Received May 26, 2009; in final form, August 3, 2009.

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or selfmodified genes. Plants are con sidered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism. Key words: metabolic engineering, transgenic plants, RNA interference DOI: 10.1134/S1068162010020020 1

INTRODUCTION

RNA interference (RNAi) is considered to be the most effective strategy for the suppression of gene expression (silencing) at the posttranscriptional stage in plants and other organisms [1].2 Since as early as the 1980s, long before the discovery of RNAi in the genetic engineering of plants, the strategy of antisense RNA (asRNA) has been used for the silencing of genes, causing the suppression of gene expression [2, 3]. Subsequently, the presence of sense and anti sense oligoribonucleotides 21–25 bp in length com plementary to the corresponding RNAs was deter mined in the cells of transgenic plants with silenced foreign genes, whereas transgenic plants with nor mally expressed foreign genes were characterized by an absence of such oligoribonucleotides [4]. Later studies demonstrated the formation of the same short interfering RNA (siRNA) duplexes 21–23 bp in length that induced the degradation of target mRNA in Drosophila [5, 6]. The mechanism of gene silencing in all eukaryotes activates several reactions in a series. Viral infection and the transgene or bidirectional transcription of mobile genetic elements results in the formation of 1 Corresponding

author; phone: +7 (4967) 330970; fax: +7 (4967) 330527; email: [email protected] 2 Abbreviations: RNAi – RNA interference; dsRNA – double stranded RNA; siRNA – small interfering RNA; asRNA – anti sense RNA.

doublestranded RNA (dsRNA). The first stage involves dsRNA slicing into 21–25 bp siRNA frag ments by a Dicer protein related to the RNAse II fa mily. The second stage is characterized by the forma tion of RISC (RNAinduced silencing complex) com plexes comprised mainly of Argonaute family proteins and siRNA. Accordingly, Argonaute family proteins are involved in the degradation of one strand of duplex siRNA, resulting in the presence of only one antisense strand of siRNA. RISC complexes combine with tar get mRNA due to the complementary interaction between siRNA and target mRNA. Argonaute de monstrates an endonuclease property (slicer) in this complex that leads to target mRNA degradation via the RNAse H mechanism [7]. Accordingly, RNA interference is a process of mRNA degradation in the presence of homologous dsRNA molecules. The most effective variant of the suppression of gene expression in plants is the transfection of hairpin structures into the cells, leading to dsRNA formation [8]. Today, the genomes of many plants have already been sequenced. According to the preliminary estima tion, the Arabidopsis genome contains 25 thousand genes encoding proteins, while the rice genome con tains 50 thousand genes [9, 10]. Now, the crucial prob lem is directed towards the identification of the func tion of each of these genes and the analysis of their interaction. To detect gene function, a disturbance in the gene activity modifying phenotype can be used. This process is known as “reverse genetics.” Previ

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THE USE OF RNA INTERFERENCE

16 : 0 Palmitic acid

Δ9Desaturase Δ12Desaturase ghSAD1 ghFAD21 18 : 0 18 : 1 18 : 2 Linoleic Oleic Stearic acid acid acid

147

18 : 3 Linolenic acid

Fig. 1. Scheme of the biosynthesis of fatty acids in cotton.

ously, antisense RNA served as an instrument in reverse genetics, while nowadays dsRNA is used. Thus, RNA interference can be assumed to be one of the methods in functional plant genomics. Currently, the strategy of RNA interference is widely used for metabolic plant engineering. Both an increase in the target metabolite synthesis and a decrease in the quan tity of objectionable substances in transgenic plants cells can be revealed under the RNAi technique. VECTOR GENE ENGINEERING CONSTRUCTS FOR RNA INTERFERENCE IN PLANTS Various genetic constructs are often used in order to suppress gene expression in plants [8, 11]. It was pre viously demonstrated that a hairpin structure was the most effective for silencing, since sense and antisense RNA form the stem of doublestranded RNA due to their complementarity, while a singlestranded RNA fragment between them forms a loop. A hairpin RNA structure obtained as a result of the transcription of these constructs was reported to contain dsRNA com prised of a minimum of 100 bp and cause effective silencing induction in plants, while the construct con taining a doublestranded fragment more than 300 bp in length is assumed to be the most effective. Hairpin structures used for RNA interference in plants have been shown to result in the suppression of the expres sion of target genes in 70–100% of transformants [12]. Several biotechnological companies have been developing specialized vector constructs for RNA interference in plants. In particular, CSIRO (Austra lia) has designed the pHANNIBAL and pKANNI BAL vectors for the transformation of dicotyledonous plants [12]. These plasmids are comprised of a 35S RNA promoter of cauliflower mosaic virus (CaMV 35S), an intron, and several sites for target gene inser tion in sense and antisense orientations at both ends of the intron. The pHANNIBAL and pKANNIBAL vectors contain bacterial genes of ampicillin and kana mycin resistance, respectively. However, a disadvan tage is the complexity of multistage gene insertion in these plasmids. Another method of gene insertion into vectors under the strategy of Gateway® recombinant cloning was designed by the American company Invitrogen [13]. The plasmids pHELLSGATE [12], pWATER RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

GATE, and pSTARGATE [14] have been constructed. The pHELLSGATE vector contains a CaMV 35S pro moter, while the pWATERGATE vector contains a lightinducible promoter of a ribulose1,5bisphos phate carboxylase RbcS small subunit. The pSTAR GATE vector has been specifically designed for the transformation of monocotyledonous plants and con tains a promoter of the maize ubiquitin gene and intron, as well as a selective marker of hygromycin resistance. The individual functions of either gene family members or genes involved in the complex bio chemical pathways can be detected. Moreover, vectors for the induced silencing of spe cific genes via RNA interference in plants have been designed using dexametazone [15]. Vectors of the pOpOff series are comprised of a pOp6 promoter and an LhGR transcription factor. Dexametazone added to media containing transformed plants was shown to activate the process of RNA interference. Such an inducible system is convenient for the detection of gene functions, since gene knockout results in the plant’s destruction. A plasmid for inducible RNA interference contain ing a phytoene desaturase gene (PDS) transformed Arabidopsis plants. A week after the placement of the plants into a dexametazonecontaining medium, the photodecoloration of the plants occurred in many transformants caused by the suppression of PDS gene expression [15]. Plasmid constructions containing heat shock gene promoters can be used for temporary gene silencing via RNA interference [16]. MODIFICATION OF THE METABOLISM OF PLANTS RESULTING IN THE IMPROVEMENT OF THEIR FOOD QUALITIES RNA interference can be used in order to obtain a plant possessing improved food qualities, for example, a modified fatty acid content. This technique has been used to obtain more qualitative cotton oil [17]. The natural oil from cotton seeds is known to contain 26% palmitic acid (C16 : 0), 15% oleic acid (C18 : 1), and 58% of linoleic acid (C18 : 2). Δ12Desaturase enzyme (FAD21) is involved in the transformation of oleic acid into polyunsaturated fatty acids (Fig. 1). The inhibition of the activity of Δ12desaturase resulted in an increase of the oleic acid content in oil Vol. 36

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up to 77%. As a result of the silencing of Δ9desaturase (SAD1), the quantity of stearic acid in cotton oil exceeded the palmitic acid content and was 40% higher compared to that in untransformed plants. The crossing of the obtained transgenic cotton plants char acterized by an inhibited expression of desaturase genes resulted in the formation of descendent plants with a high content of stearic and oleic acids in oil (up to 40%). This property was shown to be stable in posterity. Modified cotton seeds can serve as protein sources, as its content reached 22%. Thus, the cotton industry might provide the production of approximately 10 mln tons of protein per year and would be sufficient to sat isfy the demand of 500 mln people. However, cotton seeds contain a dangerous gossypol polyfunctional poison. Cotton contains gossypol as a defense against insects and pathogens. The selection of cotton free from gossypol via both the usual selection and the use of antisense RNA demonstrated no effect, since cot ton became nonresistant to infectious diseases [18]. Only recently, American researchers succeed in a reduction of the gossypol content in cotton seeds via the RNA interference technique. A construct contain ing the cadinene synthase gene (the enzyme synthesiz ing gossypol) under the control of a seedspecific pro moter was used for this aim [19]. For many diseases (cardiovascular diseases, colon cancer, and obesity), it is very important to have a diet that contains cereals with a high content of cellulose indigestible by enzymes. Starch included in cereal seeds is comprised of two polymers—amylose and amylopectin. Amylopectin chains were shown to be highly branched compared to amylose. During cook ing, amylose forms complexes stable for digestion. The GBSS (granulebound starch synthase) enzyme con trols amylose synthesis, while amylopectin synthesis is controlled by many enzymes, SBE (starchbranching enzyme) in particular. Dicotyledonous and monoco tyledonous plants differ in SBE isoforms. Potatoes possess two isoforms, SBEI and SBEII. In order to construct a potato containing more than 50% amy lose, the suppression of both SBE forms was necessary [20]. Monocotyledonous plants are characterized by the presence of three isoforms—SBEI, SBEIIa, and SBEIIb. The maize SBEIIb mutant was shown to con tain up to 50–90% amylose [21], whereas the suppres sion of both SBEI and SBEIIa revealed no effect [22, 23]. Wheat is a hexaploid cereal due to the presence of a sixfold chromosomal set. SBE genes are present in various combinations in all three genomes. RNA interference is an effective tool for the suppression of gene expression in all loci simultaneously. The simul taneous silencing of both SBEIIa and SBEIIb genes (instead of a single gene) caused an increase of the amylose content in wheat seeds by up to 70% [24]. Experiments that involved feeding rats with transgenic wheat confirmed its dietary value.

Maize with an increased lysine content due to the suppression of lysine catabolism in endosperm [25] and the subsequent insertion of the bacterial di hydrodipicolinate synthase gene CordapA from Corynebacterium glutamicum was obtained [26]. This procedure led to a 100fold enhancement in lysine synthesis in maize seeds compared to untransformed plants. To improve the food properties of potato and sweet potato and to construct tubers without amylose, sev eral research groups suppressed a few enzymes involved in starch biosynthesis via RNA interference [27, 28], while others strove to reveal plants with a high amylose content under the same method [29, 30]. As a result, plants with a high and low amylose content were obtained [31]. Manihot esculenta (Cassava) is one of the most important plants in African, South Asian, and South American countries. The main defect in this plant is its ability to accumulate cyanogenic glycosides limanarin and lotaustralin in tubers. The RNAinterference technique allowed for the suppression of cyanoglyco sides synthesis in tubers by up to 92% due to the silenc ing of the CYP79D1 and CYP79D2 genes encoding cytochromes P450 [32]. The quantity of cyanogenic glycosides in leaves of the majority of transgenic plants remained at less than 1% compared to their level in wildtype plants. Tomato plants have been obtained that were sub jected to the simultaneous or separate suppression of cellwall polygalacturonase (LePG) and expansin (LeExp1) gene activity which caused the improvement of the consistency and viscosity of tomato juice and paste [33]. RNA interference was useful for the syn thesis of tomatoes with an increased carotenoid and flavonoid content due to the inhibition of the expres sion of various genes [34–36]. The suppression of the chalcone synthase gene, on one hand, resulted in a diminished flavonoid level in tomatoes, and on the other hand, in the formation of parthenocarpic (seed less) vegetables [37]. The last technique can be useful in biotechnology for the construction of seedless fruits and berries. The majority of allergenic proteins were shown to be similar in structure and in the production of the same IgE antibodies. Since having an allergy to birch pollen antigens is usually accompanied with cross reactivity to apple proteins similar in structure, patients are obligated to limit or exclude apples from their diet. RNA interference has been used to con struct transgenic apple plants with fruits characterized by decreased allergenicity. This strategy allowed for the suppression of the synthesis of the main apple allergen Mal d1 [38]. Subsequent studies involving three patients with a cross allergy for birch pollen and apples confirmed the decreased allergenicity of these transgenic plants. These results open wide possibilities in hypoallergenic food construction.

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O 60 MT

(S)coclaurine

(S)norcoclaurine

OH NH NH 2 2

HO H3CO

(S)scoulerine

BBE

Ltyrosine

NCH3

HO

70 MT

H

HO

laudanine laudanosine codamin

H3CO (S)reticuline (R)reticuline salutaridinol CoASAc

SalAT

CoA

salutaridinol7Oacetate

H3CO O H

neopinon

H3CO codeinone

NCH3 oripavine

thebaine

morphinone

COR

codeine

COR

HO O H

NCH3

HO morphine Fig. 2. Scheme of morphine biosynthesis. 60MT, norcoclaurine6Omethyltransferase; SalAT, salutaridinol7Oacetyltrans ferase; 70MT, (R,S)reticulin7Omethyltransferase; BBE, berberine bridge enzyme; and COR, codeinone reductase.

Accordingly, the food properties of various plants can be improved via the RNAinterference technique. RNA interference is considered to be the one of the most prospective strategies of the construction of transgenic plants characterized by high biological safety due to the absence of foreign proteins. At the same time, the necessity of the analysis of a new metabolome of transgenic plants should be taken into consideration, since an undesirable quantitative shift in the synthesis of some metabolites might occur. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

ALKALOID SYNTHESIS MODIFICATIONS IN TRANSGENIC PLANTS RNA interference can be used in order to obtain a new pharmaceutical plant. Opiate poppy is a well known source of morphine and codeine formed during the majority of enzymatic reactions from Ltyrosine (Fig. 2). The codeinone reductase enzyme (COR) is responsible for codeinone transformation into codeine followed by its demethylation into morphine. Vol. 36

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RUKAVTSOVA et al. nicotine Ndemethylase

N N

CH3

N N

Nicotine

H

Nornicotine

nitrosation

N N

N O

NNitrosonornicotine

Fig. 3. Scheme of nicotine transformation into nitrosonornicotine.

NH2 N N H

N Adenine 1

PRPP PPi

AMP 2 NH3

Inosine5'phosphate 3

Xanthosine5'phosphate 4 Pi

Xanthosine 5 SAM

Xanthosine methyltransferase

SAH

7methylxanthosine 6 Rib

7methylxanthine 7 SAM

7Nmethylxanthine methyltransferase (theobromine synthase)

SAH

Theobromine 8 SAM

3,7Dimethylxanthine methyltransferase (caffeine synthase)

SAH

O H3C N O

CH3 N

N

N

CH3 Caffeine 9

RNA interference allowed for the suppression of all seven enzymes from the COR family, which resulted in the accumulation in the poppy of reticulin and its methylated derivatives—components of the drug used for malaria treatment. This appearance was unex pected, since the synthesis of such intermediates was shown to be up to eight stages higher [39]. The same research group conducted a study involving the sup pression of the synthesis of other alkaloids located in different poppy parts via antisense RNA [40] and RNA interference [41] strategies due to the inhibition of other enzymes involved in morphine biosynthesis. The use of RNA interference allowed for the con struction of a tobacco plant characterized by an increased nicotine content and an absence of carcino gens [42–44]. Tobacco’s nicotine has the ability for demethylation, leading to nornicotine formation (Fig. 3). Nornicotine is assumed to be a precursor of the Nnitrozonornicotine carcinogen. Proteins from the cytochrome P450 family are responsible for nico tine transformation into nornicotine. The suppression of one of genes of these proteins, CYP82E4, encoding nicotine demethylase via RNA interference resulted in the almost complete absence of nicotine transforma tion into nornicotine in leaves. The inhibition of another enzyme, putrescineNmethyltransferase, via RNA interference caused a decrease in the nicotine content in leaves of different lines of the tobacco plant by up to 9.1–96.7% compared to the control [45]. Japanese researchers have been trying to obtain transgenic coffee plants with caffeinefree seeds. Caf feine is synthesized from adenine during nine reac tions catalyzed by various methyltransferases (Fig. 4). The theobromine synthase gene (7Nmethylxanthine methyltransferase, CaMXMT1) encoding one of the methyltransferases involved in caffeine synthesis has been suppressed via both antisense RNA and RNA interference [46–48]. The caffeine and theobromine content in transgenic plants was shown to diminish rapidly; however, they remained different during vari ous seasons. At the same time, no harmful side sub stances were detected in the coffee seeds. FLOWERS’ COLOR MODIFICATIONS VIA RNA INTERFERENCE

Fig. 4. Scheme of caffeine biosynthesis. PRPP, phospho ribosyl pyrophosphate; PPi, pyrophosphate; SAM, Sade nosyl methionine; and SAH, Sadenosyl homocystein.

The modification of the color of flowers in trans genic plants is usually achieved by the insertion of


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sense, antisense RNAs, and interfering RNAs directly connected with the genetic determination of the bio synthesis pathways of anthocyanin pigments—dihyd roflavonol reductase [49], chalcone synthase [50–52], and chalcone isomerase [53]. A similar effect was pri marily achieved due to the expression of the antisense form of the hmg1 gene [54] indirectly involved in anthocyanin biosynthesis. This gene encodes one of the key enzymes of isoprenoid biosynthesis in the cytoplasm of plants—3hydroxy3methylglutaryl CoAreductase. The modification of the form of the color and flowers was conducted in transgenic tobacco plants with suppressed mevalonic acid synthesis. Whereas untransformed plants and plants containing the hmg1 gene in the sense orientation were character ized by a dark pink flower color, flowers of transgenic plants with an antisense gene form were reported to be pale pink. Moreover, differences in the form of the flowers’ corolla were demonstrated between trans genic and wildtype plants. The expression of the anti sense hgml gene form also resulted in male infertility of the constructed plants, which might be useful in selec tion genetic practice. Japanese researchers together with Australian ones (Suntory and Florigene companies) have designed the first blue roses [55]. To date, the usual selection failed to obtain roses with blue tinted flowers due to the majority of the red pigment. Dihydroflavonol reduc tase (DFR) is one of the genes encoding the red pig ment in roses. RNA interference has been used for gene silencing. Subsequently, the F3'5'H gene from pansies responsible for the synthesis of delphinidin blue pigment and the DFR gene from an iris have been inserted. As a result, roses with blue petals were obtained. Flowers with more intensive blue color might be obtained via the reduction of the petals’ aci dity, known to inhibit the blue pigment. Genetically modified blue roses are planned to be released by 2009 in Japan. In addition to blue roses, the blue pinks have been successfully constructed in Australia [56]. CONSTRUCTION OF TRANSGENIC PLANTS WITH A MODIFIED WOOD CONTENT The stage of lignin extraction from wood involves highpower inputs due to the technology of paper pro duction. Moreover, environmentally hazardous che mical substances are used according to this technol ogy, while lignin wastes are hardly utilized by microor ganisms. One of the important problems of contempo rary wood biotechnology involves the modification of either the quantitative or qualitative lignin content. Lignin is comprised of a mixture of polymers located in the cell walls of vascular plants. Wood from a deciduous forest contains up to 25% lignin, while a coniferous forest contains up to 35%. Biochemical lig nin and enzyme pathways and have been partially characterized [57, 58]. Lignins are derivatives of three hydroxycinnamyl alcoholspcoumaric, coniferyl, RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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and synapyl (monolignol)—differing in their me thoxylation degree. Monolignols have given rise to three lignin typesphidroxyphenyl (H), guaiacol (G), and syringic (S) (Fig. 5). In addition to the three main monolignols, lignin polymer was demonstrated to contain other phenylpropanoid compounds [59]. Dif ferent plants were characterized by the predominance of various types of lignins. The hard wood of dicotyle donous angiosperm plants mainly contains G and S lignin types, while the soft wood of gymnospermous plants is comprised of G lignins and a small number of H units [60]. The gene engineering of plants allows for manipu lation at various stages of the biosynthesis of lignin monomers. Primary experiments aimed at the modifi cation of the lignin content were mainly performed on the tobacco plants model under the strategy of anti sense RNA, cosuppression, and RNA interference. Suppression was observed of the activity of different enzymes involved in the phenylpropanoid pathway, cinnamoylCoAreductase (CCR) catalyzing trans formation of CoAderivatives of pcoumaric, caffeic, ferulic, and other acids into corresponding aldehydes [61–66]; cinnamyl alcohol dehydrogenase (CAD) responsible for aldehydes’ reduction to alcohols, the final reaction of monolignols synthesis [61–64, 66– 69]; 4coumaratCoAligase (4CL) involved in pcou marylic acid binding with acetylCoA [70, 71]; and caffeic acid Omethyltransferase (COMT) catalyzing the transfer of methyl residues to monomers [65, 68, 72]. The inhibition of these enzymes resulted in a diminished lignin content in the cells of transgenic tobacco plants, which demonstrated the prospects of this strategy for the construction of transgenic trees. One of the first studies directed toward the obtain ment of transgenic poplar hybrids with a modified lignin content was the investigation of the suppression of the activity of several enzymes involved in lignin biosynthe sis via asRNA insertion. Wood processing under the sul fate method (Kraft process) was facilitated in trees char acterized by a decreased activity of the CAD enzyme due to the improved solubilization and fragmentation of lignin caused by the modification of its structure [73–76]. Thus, the color of the wood became red or redbrown due to the accumulation of coniferyl alde hyde residues [77]. The change in the color of the wood might also be considered as an advantage in furniture production, since such wood needs no colorization. The suppression of the activity of caffeic acid O methyltransferase (CAOMT or COMT) also resulted in the modification of wood color due to a diminished Slignin content, which is considered to be disadvan tageous in the paper industry [75, 78]. A change in the caffeoylCoAOmethyltrans ferase (CCoAOMT) activity under asRNA in the cells of transgenic poplar led to a significant decrease in the lignin level by up to 60–70% as compared to untrans formed plants [57], with the same reduction in the content of both guaiacol (G) and syringic (S) lignins. Vol. 36

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HO

HO

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pcoumaric alcohol

CAD

pcoumaric aldehyde

CCR

OH

C3'H

O

OH

NH32

pcoumaroulCoA

4CL

pcoumaric acid

Phe

HO

HCT

HO

O

OH

caffeoyl alcohol

OH

CAD

caffeoyl aldehyde

CCR

caffeoylCoA

4CL

caffeic acid

OH

HO OMe

CAOMT

OH

HO

CAOMT

CCoAOMT

Glignin

coniferyl alcohol

OMe

CAD

coniferaldehyde

CCR

feruoylCoA

4CL

ferulic acid

F5H

OH

F5H

F5H

O

O CAOMT

OH

OH

4CL

5hydroxyferulic acid

OMe

HO

HO

OMe 5 hydroxyconiferyl alcohol

CAD

HO

MeO

CAOMT

CAOMT

OH

5hydroxy coniferaldehyde

CCR

HO

MeO

CAOMT CCoAOMT

CAOMT

O

OH

5hydroxyferuoylCoA

HO

HO

Slignin

OMe sinapyl alcohol

CAD

sinapoyl aldehyde

CCR

sinapoylCoA

4CL

sinapinic acid

OMe

OH

O

OH

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Fig. 5. Scheme of the phenylpropanoid pathway of the biosynthesis of lignins. 4CL, 4coumaratCoAligase; CCR, cinnamoyl CoAreductase; CAD, cinnamyl alcohol dehydrogenase; CAOMT, caffeic acid Omethyltransferase; CCoAOMT, caffeoylCoA Omethyltransferase; C3'H, pcoumaroylCoAhydroxylase; F5H, ferulat5hydroxylase; and HCT, phydroxycinnamoylCoA reductase.

The inhibition of the 4CL enzyme under cosup pression resulted in a diminished lignin content in aspen wood by up to 45% [79]. The suppression of CCR activity also decreased the lignin content; how ever, the structure of the cell walls was disturbed— modifications in the lignin–cellulose complex were demonstrated which led to the reduced growth of pop lar trees [80]. RNA interference has been used to suppress the activity of pcoumaroylCoAhydroxylase (C3'H, cytochromeP450dependent oxygenase) in the cells of a poplar hybrid [81]. A significant (up to 55%) decrease in the lignin content in the cell walls was determined. Since C3'H catalyses the stage of lignin biosynthesis common for G and S forms, a reduction in only the Glignin content was unexpected by the researchers, whereas the Slignin content in the wood remained at the previous level. Interestingly, phenyl glycosides have been accumulated in transgenic trees which can provide for a good defense in poplars against insects and fungi [82]. Such a strategy of met abolic engineering can be used to obtain biofuel, wood, and paper without sprinkling pesticides for the defense of trees against pests. In the beginning, the majority of studies of trans genic poplars were conducted in greenhouses, while subsequent ones were conducted in open soil. The main experiments have been performed by scientists from France, Belgium, Great Britain, and Sweden. It has been demonstrated that poplars with suppressed CAD or COMT synthesis cultivated over 4 years remained strong and were no different from nontrans genic poplars [76]. Wood from the trees with decreased CAD activity was shown to be more appropriate for the paper industry due to the significantly lower amount of chemicals used for delignification. The study that included the inhibition of the lac case gene lac3 in transgenic poplar plants via asRNA seems to be interesting [83]. Laccases, or pdiphe nol:O2oxydoreductases (KF 1.10.3.2), are cuprum containing glycoproteins present in many organisms, including bacteria, fungi, insects, and plants. Recently, evidence of the involvement of laccases in lignin biosynthesis has been reported based on their ability to oxidize lignin precursors (p hydroxycin namyl alcohols) and on their localization in the cell walls of xylem [84, 85]. Laccase gene inhibition had no influence on the summary growth and development of poplar trees. No changes in the lignin content or in the ratio of its forms were determined; however, one of the transgenes was characterized by a 2 to 3fold increase in the soluble phenol content in xylem and by the deformation of xylem fiber cells [83]. Most likely, RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

future geneengineering experiments using laccase genes will allow for the construction of trees with a modified wood content. There have been only a few studies conducted on coniferous plants. Plants of a Norwegian fir tree with suppressed CCR synthesis caused by the insertion of the antisense gene form have been obtained [86]. These plants differed from the nontransgenic ones only in the girth of the stem. The total lignin content was insignificantly decreased; however, the Hlignin content was lower by 34%. Recently, transgenic pine plants of Pinus radiata characterized by the diminished activity of 4couma ratCoAligase (4CL) which caused the formation of a “bonsai” dwarf phenotype were constructed [87]. The wood of transgenic trees was shown to differ in the orangebrown color and marked morphological changes in the wood structure. The lignin content in the cell walls was decreased by 50%, although mainly by the reduction in the Sform content compared to the G form. The chemical treatment of fiveyearold transgenic pines demonstrated the improvement in the wood properties necessary for the paper industry, although the decrease in the size of the trees was a side effect. Accordingly, the genetic engineering of plants via the modification of lignin biosynthesis pathways allows for the obtainment of transgenic trees with a specifically modified wood content. Such trees are planned to be cultivated in artificial forest plantations. At the same time, the possibility of negative conse quences due to the decrease in the lignin content in the wood of the plants should be taken into consideration. Lignins are known to strengthen the durability of the cell walls, to make them impermeable to water, and thus regulate the transport of water and nutrients in the vascular system. Lignins are also very important for the defense of plants against phytopathogens [88]. In conclusion, it should be noted that to date, the construction of many transgenic pants with a modified metabolism via antisense and interfering RNA tech nology has been described. The effectiveness of the synthesis of distinct metabolites frequently depends on the choice of a concrete autotroph plant. The con struction of genemodified plants for the food industry is determined by the goals of the quantitative content of the target products and distinct secondary metabo lites. The demands concerning the ease of the extrac tion of metabolites and the low cost of the obtained preparations are considered in autotroph plants of bio logically active substances for pharmaceutical use. The obtainment of biofuel and paper from the wood of Vol. 36

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