biosynthesis of defense-related proteins in transformed root cultures of trichosanthes kirilowii

Plant Physiol. (1994) 106: 1195-1204

Biosynthesis of Defense-Related Proteins in Transformed Root Cultures of Trichosanthes kirilowii

Maxim. var japonicum (Kitam.)'

Brett J. Savary and Hector E. Flores*

Graduate Program in Plant Physiology (B.J.S.) and Department of Plant Pathology and Biotechnology Institute (H.E.F.), The Pennsylvania State University, University Park, Pennsylvania 16802

We have established transformed ("hairy") root cultures from Trichosanthes kirilowii Maxim. var japonicum Kitam. (Cucurbita- ceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other root- specific defense-related plant proteins. Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacferium rhizogenes American Type Culture Collection strain 15834. Each species accumulated reproducibly a discrete protein pattern in the culture medium. Analysis of the extracellular proteins from T. kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures. Maximum protein accumulation, approaching 20 pg/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase. Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class 111 chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class 111 chitinases. The Tricho- santhes chitinases also showed reactivity with a cucumber class 111 chitinase antiserum and chitinolytic activity in a glycol chitin gel assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed 7. kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein). Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein. Roots undergoing secondary growth and differ- entiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.

Transformed ("hairy") root 'cultures have facilitated eluci- dation of root-specific metabolism in plants (Flores and Filner, 1985; Flores, 1987; Rhodes et al., 1990). Transformed root cultures stably express root-specific biosynthetic pathways and have been used to study extensively the biosynthesis of a wide range of low mol wt metabolites such as alkaloids, flavonoids, and polyacetylene and sesquiterpene phytoalex- ins (Rhodes et al., 1990; Flores and Curtis, 1992; Saito et al.,

* This work was partially supported by grants from the National Science Foundation (BCS-9 110288) and the Pennsylvania Research Corporation to H.E.F and a Sigma Xi Research Grant-In-Aid to B.J.S.

* Corresponding author; fax 1-814-863-1357.

1992). Transformed roots are obtained by inoculating plant tissues with virulent strains of the soil bacterium Agrobacte- rium rhizogenes and then isolating the adventitious roots arising from the wound sites (Tepfer, 1984). During transformation, T-DNA from the Ri (root inducing) plasmid harbored in the bacterium is inserted into the host cell genome (Chilton et al., 1982). Expression of the integrated bacterial genes affect hormone regulation in host cells, leading to induction of adventitious roots (Gelvin, 1990). Transformed root cultures generally exhibit stable and fast growth, compared to normal (untransformed) root cultures, and are genetically and biochemically stable, compared to undifferentiated cell cultures (Flores, 1987; Rhodes et al., 1990).

Plant roots also specifically synthesize and accumulate macromolecules such as storage proteins and defense-related proteins (Maeshima et al., 1985; Bowles, 1990). Examples of defense-related proteins produced in roots include proteinase inhibitors (Ryan, 1989), Pro-rich cell wall proteins (Ebener et al., 1993), and the glucanohydralases, chitinases, and /3-1,3- glucanases (Neale et al., 1990). Chitinase and 8-1,3-glucanase, also known as PRPs, are known to be coordinately induced in leaves as part of plant defensive responses to pathogenic challenge (Mauch et al., 1988a; Linthorst, 1991). Chitinase and /3- 1,3-glucanase display antifungal activities and can act synergistically (Roberts and Selitrennikoff, 1986; Schlumbaum et al., 1986; Mauch et al., 1988b). RIPS are another class of defense-related proteins produced in roots (Stirpe et al., 1992), and they inhibit protein synthesis on eukaryotic ribosomes by cleaving a specific N-glycosidic bond in 285 rRNA, preventing elongation factor 2 from binding to the 605 ribosomal subunit (Endo et al., 1987).

Storage roots from the Chinese medicinal plant Tricho- santhes kirilowii Maxim. (Cucurbitaceae) are the source of the bioactive protein TCN, a single-chain RIP (Wang et al., 1986; Maraganore et al., 1987). TCN is used to induce mid-term abortions and for treating ectopic pregnancies and tropho- blastic tumors (Tsao et al., 1986; Yeung et al., 1988). TCN was shown by McGrath et al. (1989) to inhibit human immunodeficiency virus 1 replication in human lymphocyte

Abbreviations: EAChi, extracellular acidic chitinase; EBChi, extracellular basic chitinase; IBChi, intracellular basic chitinase; MS medium, Murashige-Skoog medium; PI, isoelectric point; PRP, pathogenesis-related protein; RIP, ribosome-inactivating protein; TCN, trichosanthin.

1195

Dow

nloaded from https://academ

ic.oup.com/plphys/article/106/3/1195/6068699 by guest on 30 D

ecember 2021

1196 Savary and Flores Plant Physiol. Vol. 106, 1994

and macrophage cells in vitro, and TCN is under investigation as a potential treatment for acquired immunodeficiency syn- drome (Byers et al., 1990; Kahn et al., 1990). RIPS presumably function as defensive proteins in plants (Roberts and Seli- trennikoff, 1986; Stirpe et al., 1992), and this hypothesis is supported by in vitro antifungal activity (Roberts and Seli- trennikoff, 1986; Leah et al., 1991) and the increased fungal resistance by transgenic tobacco plants expressing a barley seed RIP (Logemann et al., 1992). To study the biosynthesis and function of bioactive defense-related proteins produced in roots, we have established transformed root cultures from several species of Trichosanthes. In this report we describe several extracellular and intracellular proteins produced in T. kirilowii var japonicum root cultures, demonstrating that class I11 chitinases are major root proteins and that TCN biosynthesis is associated developmentally with root secondary growth.

MATERIALS AND METHODS

Plant Material and Root Cultures

Seeds of Trichosanthes bracteata Voigt, Trichosanthes cucumeroides Maxim., Trichosanthes kirilowii Maxim., and T. kirilowii Maxim. var japonicum Kitam. were obtained from the Medicinal Plant Research Station (Tsukuba, Japan), and seeds of Trichosanthes cucumerina var anguina (L.) Greb. were obtained from the Institute for Genetics and Plant Tissue Cul- ture (Gatersleben, Germany). Seeds were surface sterilized in 1% sodium hypochlorite for 10 min, rinsed several times with sterile water, and germinated on basal MS medium (Mura- shige and Skoog, 1962) solidified with 0.2% Gelrite (Scott Laboratories, West Warwich, RI) in the dark. Seedlings were propagated as shoot cultures in Magenta GA-7 vessels (Sigma) containing MS medium for later infection or transferred to soil for growth in the greenhouse. Shoot cultures were kept at 24OC in a growth chamber with a 14-h light/ 10-h dark photoperiod.

One-month-old shoots were infected with Agrobacterium rhizogenes, American Type Culture Collection No. 15834, as previously described (Flores et al., 1988). Adventitious roots arising at the infection site were transferred onto MS medium supplemented with 250 pg/mL carbenicillin. Clonal lines were established as axenic cultures after several serial trans- fers of root tips to fresh media. Stock cultures were main- tained in Petri dishes by subculturing at approximately 4 weeks to MS medium (without antibiotic) and grown under low, continuous light at 24OC. Liquid batch cultures were prepared by inoculating several root tips (approximately 5 cm) into 100 mL of medium (in 250-mL Erlenmeyer flasks) or approximately 2 g fresh weight of roots from the 250-mL flasks into 2.8-L Fembach flasks containing 1 L of medium. Cultures were grown on gyratory shakers (90 rpm) under low light. Normal (nontransformed) roots were obtained by collecting adventitious roots that arose spontaneously at stem bases in shoot cultures. Storage and enlarging fibrous roots were collected from greenhouse-grown plants.

Preparation of Extracts from Root Cultures

Root extracts were prepared by homogenizing 0.1 g of lyophilized tissue in 2.5 mL of ice-cold extraction buffer (25

m NaP04, pH 7.0, with 250 mM NaC1, 10 ~ I V I EDTA, 10 m~ thiourea. 5 mM DTT, 1 mM PMSF, and 1.5% polyvinyl- polypyrolydone). One milliliter was recovered after vacuum filtration through glass microfiber filters and cenlrifuged, and the total protein in the supematant was precipitated by addition of 6 mL of cold acetone (-17OC). After the sample was centrifuged, the pellets were dissolved in 2 inL of 20 mM NaP04 (pH 7.0) containing 20 mM EDTA and 5 mM DTT; insoluble material was removed by recentrifugation. CM- Accell SepPaks (Waters, Milford, MA) were equilibrated with 10 mL of 20 mM NaP04 buffer (pH 7.0), samples were loaded, and the flow-through containing unretained (ad dic) proteins was collected. After the SepPak was washed with 3 mL of loading buffer (which was pooled with the flow-through), retained (basic) proteins were eluted with 3 ml, of 250 m~ NaCl in 20 mM NaP04 buffer (pH 7.0). Dilute protein solu- tions were concentrated using TCA precipita tion by the method of Peterson (1977). Briefly, to each I-mL sample containing 5 to 100 pg protein/ml, 100 pL of Na deoxycholate was added, mixed, and incubated for 10 min. ‘Then 100 pL of 72% TFA were added, mixed, and incubated on ice for 15 min and then centrifuged for 8 min. The supernatants were immediately removed, and the pellets were washed three times with ice-cold 80% acetone. Pellets were then dissolved in SDS-PAGE buffer (Laemmli, 1970). Protein determinations were done using the reagent of Bradford (1976) for total proteins and by using an LKB Ultrascan XL laser densitometer for individual proteins; peak areas were calculated by signal integration. BSA (Pierce, Rockford, IL) was usell as the protein standard.

Extracellular proteins in 1 mL of culture medium were prepared for SDS-PAGE analysis by precipitaíion with Na deoxycholate and TCA as described for intracellular proteins. For preparative analyses, proteins in media were concentrated by ultrafiltration (1 0,000 NMWL membrane, Millipore), then precipitated with ammonium sulfate (80% final saturation), and dissolved in and dialyzed against 20 m PJaP04 buffer (pH 7.0).

Protein Purification and Amino Acid Analysis

Ion-exchange chromatography was performed with Milli- pore CM- and DEAE-MemSep membrane cartridges according to the manufacturer’s instructions. Final purifications from fractions isolated from MemSep cartridges or SepPaks were obtained by reverse-phase HPLC with a Macrosphere 300-C8 column (Alltech, Deerfield, IL) using a 60-min linear solvent gradient of 15 to 60% acetonitrile in 0.1% aqueous TFA at a flow rate of 1 mL/min. Purity of separated proteins was determined by SDS-PAGE and silver staining. Amino acid compositions and N-terminal sequences of rotein ins were determined with an Applied Biosystems (Foster City, CA) 420A/H analyzer and 477A sequencer, respectively.

Electrophoresis and Western Blot Analysis

SDS-PAGE was performed with 13.5 or 15% acrylamide discontinuous gels (Laemmli, 1970) using a Mini-Protein I1 electrophoresis cell (Bio-Rad, Richmond, CA) according to the manufacturer’s instructions. Sigma low-mol wt protein

Dow



ecember 2021

Defense Proteins from Jrichosanthes Root Cultures 1197

markers were used. Proteins were stained with Coomassieblue R-250 or silver stain (Sigma).

Electroblotting of proteins from gels to an Immobilon-Ppolyvinylidene difluoride membrane (Millipore) was per-formed with a Bio-Rad Mini-Trans electrotransfer cell, for 1h at 125 V (constant voltage), using 10 mM 3-(cyclohexylam-ino)propanesulfonic acid (pH 11.0 with NaOH) and 10%methanol transfer buffer (LeGendre and Matsudaira, 1989).Membranes were developed with the Promega secondaryantibody-alkaline phosphatase detection system according tothe manufacturer's instructions. A goat anri-TCN serum ob-tained as a protein-A-purified preparation from Dr. MichaelPiatak (GeneLabs, Redwood City, CA) was further affinitypurified using TCN immobilized on polyvinylidene difluoridemembrane following SDS-PAGE (Sambrook et al., 1989). Arabbit anti-cucumber chitinase serum (received from Dr. JohnRyals, Ciba-Geigy Corp., Research Triangle Park, NC) and achicken anti-osmotin serum (received from Dr. Ray Bressan,Purdue University, West Lafayette, IN) were used as received.

Chitinase Activity Gel Assays

Chitinolytic activities were detected using the glycol chitingel assay of Trudel and Asselin (1989). Proteins resolved on15% SDS-polyacrylamide gels containing 0.2% glycol chitin(w/v) (Sigma) were renatured using two 15-min washes of

25% isopropanol in 100 mm sodium acetate (pH 5.5) andthen incubated overnight at 37°C in 100 mvi sodium acetatebuffer without isopropanol. Gels were then stained with0.01% calcofluor white M2R (Sigma) in 0.5 M Tris (pH 9.0)for 5 min, destained in water, and observed under long-wavelength UV. Chitinolytic activity was visualized ascleared areas against a fluorescent background.

RESULTS

Establishment of /4groftacferium-Transformed RootCultures

We isolated adventitious roots from plantlets infected withAgrobacterium rhizogenes American Type Culture Collectionstrain 15834 and established 12 transformed root cultures.These included clones from Trichosanthes bracteata, Tricho-santhes cucumerina var anguina, Trichosanthes cucumeroides,Trichosanthes kirilowii, and T. kirilowii var japonicum. Repre-sentative root clones for three Trichosanthes species and stor-age roots from a greenhouse-grown T. kirilowii var japonicumplant are shown in Figure 1. Transformed roots displayed arigid, fibrous root phenotype with a characteristic primarygrowth anatomical appearance. All clones exhibited stablephenotypes and fast growth through 4 years in culture.Maximum yields of total extracellular proteins produced by

Figure 1. Comparison of J. kirilowii var japonicum storage root morphology with that of transformed root cultures fromthree other Jrichosanthes species. A, Storage roots of T. kirilowii var japonicum from plant grown in pot. Transformedroot cultures of T. kirilowii var /apon/cum (B), T. cucumerina var anguina (C), and T. cucumeroides (D) are shown. Rootcultures were established as described in "Materials and Methods" and grown for 25 to 30 d.

Dow



ecember 2021


root cultures typically ranged from 10 and 50 mg/L medium.Trichosanthes root cultures produced species-specific patternsof extracellular proteins that included both common andunique major proteins (Fig. 2).

Root Growth and Extracellular Protein AccumulationPatterns

A representative growth curve prepared for a T. kirilowiivar japonicum root clone is shown in Figure 3A. The rootculture grew rapidly (doubling time approximately 3 d),reaching stationary phase at approximately d 25. Maximumtissue accumulation under these conditions was approxi-mately 200 g fresh weight/L medium, representing about a1000-fold increase in biomass starring from a single root tipinoculum. Extracellular proteins accumulated in the culturemedium during the time course were also examined by SDS-PAGE (Fig. 3B). Two sets of abundant proteins were ob-served. The first set was accumulated transiently during theexponential growth phase (d 15-25) and included five bands23 to 36 kD in size. The second set, which included a 25-kDprotein and two proteins of approximately 36 kD, beganaccumulating during late exponential growth but persistedthrough stationary growth phase. The accumulation patternsfor individual proteins in root cultures were found to beconsistent and reproducible between replicates (data notshown). Densitometric determinations for selected proteinsare shown in Figure 3C. Maximum protein accumulationoccurred before the end of exponential phase at approxi-mately d 20, whereas the early set of proteins were stillpresent, and corresponded to a total protein yield of approx-imately 20 /ig/mL.

Differential Protein Accumulation in Culture Medium andRoot Tissue

To facilitate screening and electrophoretic analyses of pro-tein extracts, a rapid cation-exchange chromatography sepa-

66kD-45kD-36 kD-29 kD-24 kD20 kD-

14 kD-

Figure 2. Patterns of extracellular proteins accumulated in the rootculture medium of three Trichosanthes species. MWM, Molecularmass markers (sizes are as indicated); TKJ, T. kirilowii var japonicum;TCA, T. cucumerina var anguina; TCu, 7". cucumeroides. Samples forelectrophoresis were prepared and processed as described in "Ma-terials and Methods." About 15 ^g of protein were loaded in thelanes for each species.

IB;2 1.0o>iS o.i

20 30Sample Day

-20 kD

-14 kD

10 15 20 25 30

Sample Day

Late set--•--37.5 kD--*--36kD--•--24 kD

20 30Sample day

Figure 3. Time course of root growth and extracellular proteinaccumulation in the medium of T. kirilowii var /apon/'cum-trans-formed root cultures. A, Growth curve of fresh and dry weightaccumulation over 45 d. B, SDS-PACE of total culture mediumproteins. MWM, Molecular mass markers. C, Densitometric deter-mination of selected individual proteins accumulated in the culturemedium. Root cultures were prepared as described in "Materialsand Methods" and grown under continuous light (50 /imol m~2 s"1).Flasks were harvested in triplicate every 5 d. Each sample representsproteins precipitated from 1 ml of medium (as described in "Ma-terials and Methods").

ration was developed using Waters' CM-Accell SepPaks.Extracellular and intracellular proteins extracted from a T.kirilowii var japonicum root culture were separated into acidicand basic fractions and resolved by SDS-PAGE (Fig. 4). Theelectrophoretic protein pattern observed for the extracellularextract was clearly distinguished from that observed for tissue(intracellular) extracts, suggesting that the major proteinsoccurring in the culture medium were secreted from roots

Dow



ecember 2021

Defense Proteins from Trichosanthes Root Cultures 1199

Extracellular Intracellular

-•-66kD

-»-36kD-*-29kD-«-24kD-«-20kD

-«-14kD

Figure 4. Differential accumulation of acidic and basic proteins inthe medium and tissues of a T. kirilowii var japonicum root culture.Protein fractions were separated using a Waters CM-Accell SepPak(cation exchanger) and resolved by SDS-PAGE. Basic proteins weredefined as those retained in the cation-exchange cartridge in 20nriM NaPO4 buffer at pH 7.0. Lanes 1 to 3 represent total, acidic,and basic extracellular proteins concentrated from the culture me-dium, respectively. Lanes 5 to 7 represent total, acidic, and basicintracellular proteins extracted from root tissue, respectively. Lane4, Molecular mass markers (MWM; sizes are indicated to the rightedge of gel).

rather than being released from damaged cells. Among theextracellular proteins, two basic proteins (lane 3) of 24 and26 kD were found to correspond with proteins that accumu-lated transiently during the growth time-course experiment(Fig. 3B). A 25-kD protein, not retained on the SepPak as anacidic protein (Fig. 4, lane 2), corresponded with those thataccumulated stably during stationary growth phase (Fig. 2B).Protein recovery determinations showed that basic proteinscontributed from about 40% to less than 15% of total proteinas cultures advanced into stationary phase (data not shown),consistent with the accumulation pattern observed in Figure3 for individual basic proteins. Separation of intracellularproteins (Fig. 4, lanes 5-7) resulted in about 30% of theprotein being recovered as basic proteins. Major intracellularbasic proteins observed included a 26-kD protein and a seriesof 32- to 34-kD proteins.

Identification of Major Root Proteins as Class IIIChitinases

TCN is reported to be a small basic protein (pi 9.4) ofapproximately 24 to 26 kD (Wang et al., 1986; Maraganoreet al., 1987). Three major extracellular proteins and oneintracellular protein produced by the T. kirilowii var japoni-cum root clone, which were similar in size to TCN, werepurified by reverse-phase HPLC and further analyzed todetermine any potential relationship with TCN. Partial N-terminal amino acid sequences and amino acid compositionswere obtained for each and are shown in Figure 5 and TableI, respectively. Three of these proteins displayed a highsequence and composition homology with each other andwith class HI chitinases from other species (Shinshi et al.,1990). These included a 25-kD EAChi, a 24-kD EBChi, anda 26-kD IBChi. EAChi was further analyzed to corroborate

its identity as a class III chitinase. Figure 6A shows SDS-PAGE of EAChi, total extracellular proteins, and TCN. Thewestern blot showed that EAChi was highly immunoreactivewith cucumber class III chitinase antiserum (Fig. 6B), dem-onstrating a serological relationship with cucumber chitinase,consistent with its amino acid sequence and compositionhomology with cucumber chitinase. The other two chitinasespurified from the root culture were also reactive with theantiserum, as were two additional unpurified proteins, in-cluding one observed in the medium (22.5 kD) and anotherin root tissue (25 kD) (data not shown). EAChi also displayedchitinolytic activity in the glycol chitin gel assay (Trudel andAsselin, 1989) (Fig. 6C), confirming its identity as a chitinase.A control gel stained without renaturation of proteins did notproduce clearing or quenching of fluorescence in gels. Addi-tional chitinolytic activity, which corresponded with proteinsof approximately 35 kD, was detected in the glycol chitin gelassay, but these proteins showed no reactivity with thecucumber chitinase antiserum, suggesting that they are notclass III chitinases.

A 26-kD extracellular basic protein was also purified andsequenced (Fig. 5). This protein showed no relationship tochitinases or to TCN but did have some sequence homologywith osmotin, a member of the group 5 family of PRPs nowreferred to as permatins, which are known to accumulateconstitutively in tobacco roots (Singh et al., 1987; Neale etal., 1990; Linthorst, 1991; Vigers et al., 1991). The tentativeidentification of this protein as a permatin is supported by itsstrong reactivity with an osmotin antiserum in a western blotassay (data not shown). This protein was observed to accu-mulate transiently in the culture medium (Fig. 3) and appearsto be common to all Trichosanthes species (Fig. 2).

Intracellular Protein Patterns and TCN Biosynthesis inDifferent Root Types of T. kirilowii var japonicum

We examined intracellular proteins in different T. kirilowiivar japonicum root types for their ability to produce TCN.TCN is reported to be a major basic protein in storage roots,accounting for up to 30% total extractable protein (Wang etal., 1986). The basic proteins obtained using CM-Accell

ATrichosantftes, EAChiTrichosanlhes, EBChiTrichosanthes,\BChiCucumber, acidicArabidopsis, basicTobacco, acidicTabacco, basic

BTrictwxanthes, 24kDextracellular basic proteinOsmotinTobacco PR-5Trichosanthin

(N- terminus)- -<C- terminus)A GA G

A G GA GG GG D

A G O

AAHAj^V

Y W G Q N G N E G T L A S TY W G Q N Y N A G H L SR W G Q N G N E G S L S A TY W G Q N G N E G S L A S TY W G Q N G N E G N L S A TY W G Q N G N E G S L A D T

V V Y W G Q D V G E G K L I D T

A T F T Y K N - - P - T I WA T I E V R N N C P Y T V WA T F D I V N K C T Y T V WD V S F R L S D A N S K S Y R K F I T

Figure 5. Comparison of the partial N-terminal amino acid se-quences from Trichosanthes root proteins with class III chitinases(A) and osmotin and TCN (trichosanthin, B). Deduced amino acidsequences were obtained from nucleotide sequences reported forcucumber (Metraux et al., 1989), Arabidopsis (Samac et al., 1990),tobacco (Lawton et al., 1992), and osmotin (Singh et al., 1987). TCNsequence is from Maraganore et al. (1987). Hyphens representunassigned peaks from protein sequencer.

Dow



ecember 2021


Table I. Amino acid compositions of class III chitinases from Trichosanthes and other speciesAmino acid compositions for cucumber, tobacco, and Arabidopsis chitinases were determined

from DMA sequences (Metraux et al., 1989; Lawton et al., 1992; Samac et al., 1990, respectively).

Amino Acid

AsxGlxSerClyHisArgThrAlaProTyrValMetlieLeuPheLysCysTrp

Acidic Proteins—————————— ———— —— —Cucumber

37183129

223

30138

143

152313967

Tobacco

4322193324

10261213162

132411868

EAChi'

39102432079

27206947

20144

——

Arabidopsis

341526294

101321141211

31424131568

Basic Proteins

Tobacco

2824213049

10171312144

1627131576

IBChi'

271718386

128

23146

1029

181111——

EBChi'

351622240

106

28146

102

1026169

——

* Trichosanthes root proteins: EAChi and EBChi were purified from culture medium and IBChi waspurified from root tissue.

SepPaks were resolved by SDS-PAGE and examined withthe TCN antiserum (Fig. 7). We found differences in proteinand antigen patterns between fibrous roots (both transformedand normal) and storage roots that we attribute to differencesin root development phenotypes. The SDS-PAGE proteinpatterns (Fig. 7A) from fibrous roots (transformed and nor-mal, lanes 1 and 2, respectively) showed distinct differencesfrom that observed for storage roots (lane 3). Enlargingfibrous roots (lane 4) exhibited a protein pattern more similarto the mature storage roots, indicating a change in proteinaccumulation as roots initiated secondary growth. Both fi-brous and storage roots accumulated small basic proteins ofthe size reported for authentic TCN (24-26 kD), but thewestern blot revealed differences in protein immunoreactiv-

ities with the TCN antiserum (Fig. 7B). In fibrous roots onlyproteins of the size range 32 to 35 kD were immunoreactive(lanes 1 and 2), whereas in the storage root strong antigenreactivity was observed for two smaller proteins at 24 and 30kD (lane 3). The 24-kD antigen in storage roots was purifiedto homogeneity, and its amino acid composition matchedthat of authentic TCN (data not shown). The 30-kD antigenicprotein is assumed to be karasurin, a TCN isoform previouslyreported in T. kirilowii var japonicum (Toyokawa et al., 1991).The enlarging fibrous roots (lane 4) also displayed a stronglyreacting band matching authentic TCN but showed muchreduced antigenic activity for 32- to 35-kD proteins. Follow-ing longer color development time of western blots in otherexperiments, an immunoreactive band corresponding in size

Figure 6. Analysis of EAChi purified from J.kirilowii var/apon/'cum root culture medium. A,Proteins visualized with Coomassie blue R-250stain following SDS-PACE. B, Western blot of aduplicate gel using the cucumber class III chi-tinase antiserum. C, Detection of chitinolyticactivity in a glycol chitin gel assay. TEP, Totalextracellular proteins; MWM, molecular massmarkers.

B

35 kO

24 kD

35 kD 35 kD

24 kD

TCN TEP EAChi MWM

TCN TEP EAChi MWM TCN TEP EAChi MWM

Dow



ecember 2021


I _ . fCO C (B k.c c "•! a5 O O •£1= Z M uj

45kD-35kD-

29 kD-24 kD-

20 kD-«-

B

-«-TCN

45kD-35kD-

29 kD-24 kD- -•-TCN

Figure 7. Intracellular protein patterns of basic proteins accumu-lated in different T. kirilowii var japonicum root phenotypes. A,Coomassie blue R-250 staining of proteins resolved by SDS-PACE.B, Western blot using a TCN antiserum. Fibrous (primary growth)root is represented by transformed and normal roots, and secondarygrowth root is represented by a fully developed storage root andenlarging fibrous roots. The duplicate gel for electroblotting wasprepared with approximately 10-fold less protein than that loadedon the Coomassie blue R-250-stained gel.

to TCN was observed in fibrous root extracts (not shown).No extracellular proteins were immunoreactive with the TCNantiserum.

The antiserum used in Figure 7 was refined using SDS-PAGE immunoaffinity purification to obtain monspecific an-tibodies (Sambrook et al., 1989). This resulted in increasedsensitivity for TCN and eliminated the immunoreactivityobserved for the 32- to 35-kD proteins produced in fibrousroots. To establish that T. kirilowii vary'aponicum-transformedroots produced low levels of TCN, we resolved a root extractusing pH gradient elution on an SCX HPLC column (Alltech).A partially pure 24-kD protein was enriched in a fraction,approximately pH 9.3, and was observed to be immunoreac-tive with the monospecific TCN antiserum and to co-migratewith TCN isolated from storage roots (Fig. 8). TCN from T.kirilowii var japonicum migrated slightly slower than TCNfrom T. kirilowii, indicating a small difference in TCN sizebetween the two genotypes. TCN was calculated to representapproximately 0.5% of total soluble protein in fibrous trans-formed root tissue. Evidence suggesting that TCN producedin the T. kirilowii var japonicum-transformed roots is activewas the dramatic protein synthesis inhibition in the rabbitreticulocyte lysate mRNA translation assay (Lee-Huang et al.,1991) by a total root protein extract added at concentrationsof 1 to 10 /ig protein extract/mL. Pure RIPs are typicallyactive in the nanogram range in this assay, and TCN as 0.5%of total soluble protein in fibrous root tissue would accountfor the observed activity.

DISCUSSION

We established transformed root clones in the Trichosanthesspecies tested by infecting plantlets with A. rhizogenes Amer-ican Type Culture Collection strain 15834. These roots dis-played the morphology commonly observed for root cultures(Tepfer, 1984; Flores and Filner, 1985; Gelvin, 1990; Rhodeset al., 1990), that is fibrous roots with primary anatomyincluding a well-developed cortex surrounding a central vas-cular cylinder, with new tissues developing from an apicalmeristem. In contrast to the typical hairy root morphologyobserved for most other genera, Trichosanthes root cloneswere relatively much larger in diameter (occasionally ap-proaching 1 cm in diameter) and rigid and showed low root-hair densities. Enlarged and "fleshy* phenotypes were ob-served in some Trichosanthes root clones, as seen in theforeground of a T. cucumerina var anguina culture and aT. cucumeroides clone (Fig. 1, B-C), and are suggestive ofstorage roots. The development of T. kirilowii var japonicumstorage roots in potted plants appeared similar to that ofIpomoea batatas (sweet potato), in which secondary growthoccurs through radial enlargement by cell division alongdispersed anomalous cambia within the vascular cylinder(Esau, 1977; Sirju-Charran and Wickham, 1988). However,enlargement of Trichosanthes fleshy cultured roots was dueprincipally to cortical cell expansion rather than true second-ary growth (B.J. Savary and H.E. Flores, unpublished obser-vations). Most Trichosanthes root clones were observed tobecome intensely green when grown in direct light, suggest-ing chloroplast development. Transformed roots in otherspecies have demonstrated the ability to become photosyn-thetic and photoautotrophic (Flores et al., 1993).

Root phenotype was found to be very important in TCNproduction in T. kirilowii var japonicum roots. TCN wasaccumulated only at low levels (approximately 0.5% of totalsoluble proteins) in transformed and normal fibrous roots,

5 storage root| TCN, TCN,

TK TKJ

1 2 3 4

Figure 8. Western blot of T. kirilowii var /apon/'cum-transformedroot extracts. The membrane was developed using a monospecificTCN antiserum preparation (see "Materials and Methods"). Lane 1,TCN semipurified from transformed root tissues; lane 2, 50 ng ofTCN obtained from 1. kirilowii (TK); lane 3, 100 ng of TCN obtainedfrom J. kirilowii (TK); lane 4, TCN purified from T. kirilowii varjaponicum (TK)) storage roots.

Dow



ecember 2021


but upon secondary growth induction leading to storage root formation, protein production pattems shifted and TCN was accumulated as the major soluble protein (>20% total soluble proteins) (Fig. 7). Furthermore, these changes in protein pattems appear to occur early during initiation of storage root development from fibrous roots. Similar changes in root protein expression pattems associated with developmental events have been reported in other species (Maeshima et al., 1985; Ebener et al., 1993). The common pattems observed for normal and tránsformed roots also indicate that Ri-plas- nud insertion in the transformed roots does not affect the accumulation of the major proteins.

Our results indicate that Trichosanthes roots may produce as many as five class I11 chitinases, including three in the culture medium and two intracellularly. The three purified in this study showed a remarkable conservation of N-terminal amino acid sequences, in comparison with chitinases char- acterized in other species (Fig. 5 and Table I), but are distinct proteins based on their differences in relative PI, size, extra- or intracellular location, N-terminal amino acid sequences, and amino acid compositions (Fig. 5 and Table I). Class I11 chitinases generally occur as single-copy or small gene families in other species reported to produce them (Samac et al., 1990; Lawton et al., 1992, 1994). Further biochemical and genetic characterization of these proteins could provide in- formation conceming their regulation in roots and the evo- lution of class I11 chitinases. Trichosanthes roots also appear to produce other classes of chitinases. The extracellular 32- to 35-kD proteins showed strong chitinolytic activity in glycol chitin gel assays but were not reactive with the cucumber class I11 chitinase antiserum (Fig. 6). Class I11 chitinases, such as that represented by the cucumber protein, show no sequence homology with the conserved catalytic regions of class I and I1 chitinases and do not react with antisera raised against the latter classes (Metraux et al., 1989; Shinshi et al., 1990). The presence of multiple chitinase classes, in addition to class 111, has been demonstrated in other species, including tobacco (Lawton et al., 1992), Arabidopsis (Samac et al., 1990), and chickpea (Vogelsang and Barz, 1993). Two new classes of chitinases have been reported recently: class IV chitinases, which are structurally homologous to the class I proteins (Collinge et al., 1993), and class V chitinases, which may be evolutionarily derived from bacterial exo-chitinases, similar to class I11 chitinase (Watanabe et al., 1992; Melchers et al., 1994).

Using Waters' Accell SepPaks, we developed a rapid and convenient method to separate acidic from basic proteins in both medium concentrates and tissue extracts (Fig. 4). We have found that acidic or basic proteins can also be directly concentrated from culture media on the anion or cation exchanger, respectively, following appropriate pH adjust- ment and filtration. Protein accumulation in culture media appears to occur directly through secretory processes rather than stable accumulation of intracellular proteins that are released through cell lysis. This is supported by the distinct and differential accumulation of several species of major proteins, including the chitinases that were distinguished by size and relative PI, between the medium and root tissues. The acidic class I11 chitinase from cucumber was shown to specifically accumulate extracellularly (Metraux et al., 1989),

and all genes described for this class contain I\T-terminal secretory peptide sequences.

Extracellular Trichosanthes root proteins were accumulated in culture media in species-specific (Fig. 2) and growth phase- specific patterns (Fig. 3). These likely represent normal expression pattems in vivo. Basic isoforms of chitinase and other PRPs are known to accumulate developmentally in tobacco roots (Neale et al., 1990; Linthorst, 1991), and the inducible chitinase gene from cucumber was also recently shown to be expressed developmentally in roots (IMetraux et al., 1989; Lawton et al., 1994). Secretion of PRPs in Tricho- santhes roots presumably functions to protect juvenile roots against soil fungal pathogens. In preliminary experiments the additive combination of extracellular acidic and basic protein fractions, which contained a chitinase and putative permatin, respectively, greatly enhanced antifungal activity compared to activity exhibited by the individual fractions (I3.J. Savary and H.E. Flores, unpublished data). Synergistic antifungal activities of PRPs have been reported previously (Roberts and Selitrennikoff, 1986; Mauch et al., 1988b; Leah et al., 1991). TCN presumably functions as a defense-related protein in Trichosanthes root tissues and may act in ccncert with the extracellular PRPs. The intracellularly accumulated TCN may be released into the rhizosphere upon cell lysis due to fungal pathogens or insect herbivory, whereupon it can interact synergistically with extracellular PRPs to provide enhanced antifungal or insect deterrent activities. RIPS have been shown to exhibit antifungal activity in vitro (Roberts and Selitrennikoff, 1986; Leah et al., 1991) and to act synergistically in antifungal activity when combined with hydro- lytic PRPs (Leah et al., 1991).

Trichosanthes root cultures may be useful in further studies of biosynthesis and function of root-specific defense-related proteins. Determining conditions for in vitro induction of storage roots will expedite molecular dissection of storage root development and TCN biosynthesis. This system will also facilitate further investigation of PRP expression in roots. Additionally, Trichosanthes-transformed roots can be used to model scaled-up production of bioactive proteins in root cultures. The distinctive morphology displayed by Tricho- santhes-transformed roots may facilitate their growth in root bioreactors because they appear to be less sensitive to shear stress, and they are likely to show reduced flow resistance in submerged culture and reduced capillary hold-up in tickle- bed reactors (Ramakrishnan and Curtis, 1993). In small-scale trials, we have observed that extracellular protein pattems in liquid-dispersed spray/trickle-bed reactors are simjlar to that observed in flask-batch cultures (Fig. 3B). Pilot xale trials using trickle-bed and bubble column reactors are in progress to determine whether these changes in extracellular protein pattems can be used to monitor root growth in larger-scale reactor configurations.

ACKNOWLEDGMENTS

TCN (GLQ223) and anti-TCN antiserum were gift!i from Dr. Michael Piatak, GeneLabs Inc., the cucumber class 111 chitinase and the antiserum for it were gifts from Dr. John Ryals, Ciba-Geigy, and an osmotin antiserum was a gift from Dr. Ray Bressiin, Purdue University. Testing of root extracts for RIP activity was kindly performed by Dr. Sylvia Lee-Huang, New York University Medical

Dow



ecember 2021


School. We thank Paula Michaels for assisting with plant transfor- mations and Robert Boor (Protein and Nucleic Acid Analysis Facility, Pennsylvania State Biotechnology Institute) for amino acid composition and sequence analyses.

Received May 26, 1994; accepted July 26, 1994. Copyright Clearance Center: 0032-0889/94/106/1195/10.

LITERATURE CITED

Bowles DJ (1990) Defense-related proteins in higher plants. Annu Rev Biochem 5 9 873-907

Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Anal Biochem 7 2 248-254

Byers VS, Levin AS, Waites LA, Starrett BA, Mayer RA, Clegg JA, Price MR, Robins RA, Delaney M, Baldwin RW (1990) A phase 1/11 study of trichosanthin treatment of HIV disease. AIDS

Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3 31-40

Chilton M-D, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J (1982) Agrobncterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature 295 432-434

Ebener W, Fowler TJ, Suzuki H, Shaver J, Tierney ML (1993) Expression of DcPRPl is linked to carrot storage root formation and is induced by wounding and auxin treatment. Plant Physiol

Endo Y, Mitui K, Motizuki M, Tsurugi K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28s ribosomal RNA caused by the toxins. J Biol Chem 262 5908-5912

Esau K (1977) Anatomy of Seed Plants, Ed 2. John Wiley & Sons, New York

Flores HE (1987) Use of plant cells and organ cultures in the production of biological chemicals. In RO Mumma, H Lebaron, RC Honeycutt, JH Duesing, eds, Applications of Biotechnology to Agricultural Chemistry. American Chemical Society Sympo- sium Series, Vol 334, Washington, DC, pp 66-86

Flores HE, Curtis WR (1992) Approaches to understanding and manipulating the biosynthetic potential of plant roots. Ann NY Acad Sci 665: 188-209

Flores HE, Dai Y-R, Cuello JL, Maldonado-Mendoza IE, Loyola- Vargas VM (1993) Green roots. Photosynthesis and photoautotro- phy in an underground plant organ. Plant Physiol 101: 363-371

Flores HE, Filner P (1985) Metabolic relationships of putrescine, GABA, and alkaloids in cell and root cultures of Solanaceae. In K- H Neumann, W Barz, E Reihard, eds, Primary and Secondary Metabolism of Plant Cell Cultures. Springer-Verlag, New York,

Flores HE, Pickard JJ, Hoy MW (1988) Production of polyacetylenes and thiophenes in heterotrophic and photosynthetic root cultures of Asteraceae. In J Lam, H Breteler, T Amason, L Hansen, eds, Chemistry and Biology of Naturally-Occurring Acetylenes and Related Compounds. Elsevier, New York, pp 233-254

Gelvin SB (1990) Crown gall disease and hairy root disease, a sledgehammer and a tackhammer. Plant Physiol92 281-285

Kahn JO, Kaplan LD, Gambertoglio JG, Bredesen D, Arri CJ, Turin L, Kibort T, Williams RL, Lifson JD, Volberding PA (1990) The safety and pharmacokinetics of GLQ223 in subjects with AIDS and AIDS-related complex: a phase I study. AIDS 4

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685

Lawton KA, Beck J, Potter S, Ward E, Ryals J (1994) Regulation of cucumber class 111 chitinase gene expression. Mol Plant-Microbe Interact 7: 48-57

Lawton K, Ward E, Payne G, Moyer M, Ryals J (1992) Acidic and basic class 111 chitinase mRNA accumulation in response to TMV infection of tobacco. Plant Mol Biol 1 9 735-742

Leah R, Tommerup H, Svendsen I, Mundy J (1991) Biochemical

4 1189-1196

101: 259-265

pp 174-185

1197-1204

and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem 266: 1564-1573

Lee-Huang S, Huang PL, Kung H-F, Li B-Q, Huang PL, Huang P, Huang HI, Chen H-C (1991) TAP 29: an anti-human in”uno- deficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells. Proc Natl Acad Sci USA 88: 6570-6574

LeGendre N, Matsudaira PT (1989) Purification of proteins and peptides by SDS-PAGE. In PT Matsudaira, ed, A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, New York, pp 49-69

Linthorst HJTvI (1991) Pathogenesis-related proteins of plants. CRC Crit Rev Plant Sci 1 0 123-150

Logemann J, Jach G, Tommerup H, Mundy J, Schell J (1992) Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants. Bio/Tech- nology 1 0 305-308

Maeshima M, Sasaki T, Asahi T (1985) Characterization of major proeins in sweet potato tuberous roots. Phytochemistry 24

Maraganore JM, Joseph J, Bailey MC (1987) Purification and characterization of trichosanthin: homology to the ricin A chain and implications as to mechanism of abortifacient activity. J Biol Chem

Mauch F, Hadwiger LA, Boller T (1988a) Antifungal hydrolases in pea tissue. I. Purification and characterization of two chitinases and two P-1,3-glucanases differentially regulated during development and in response to fungal infection. Plant Physiol 87:

Mauch F, Mauch-Mani B, Boller T (1988b) Antifungal hydrolases in pea tissue. 11. Inhibition of fungal growth by combination of chitinase and @-1,3-glucanses. Plant Physiol88: 936-942

McGrath MS, Hwang KM, Caldwell SE, Gaston I, Luk K-C, Wu P, Ng VL, Crowe S, Daniels J, Marsh J, Deinhart T, Lekas PV, Vennari JC, Yeung H-W, Lifson LD (1989) GLQ223: an inhibitor of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phago- cyte lineage. Proc Natl Acad Sci USA 86: 2844-2848

Melchers LS, Apotheker-de Groot M, van der Knaap JA, Ponstein AS, Sela-Buurlage MB, Bo1 JF, Cornelissen BJC, van den Elzen PJM, Linthorst HJM (1994) A new class of tobacco chitinases displays antifungal activity. Plant J 5 469-480

Metraux JP, Burkhart W, Moyer M, Dincher S, Middlesteadt W, Williams S, Payne G, Carnes M, Ryals J (1989) Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proc Natl Acid Sci USA

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tissue cultures. Physiol Plant 1 5 473-497

Neale AD, Wahleithner JA, Lund M, Bonnett HT, Kelly A, Meeks- Wagner DR, Peacock WJ, Dennis ES (1990) Chitinase, j3-1,3- glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation. Plant Cell 2 673-684

Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 8 3

Ramakrishnan D, Curtis WC (1993) Fluid dynamics on plant root cultures for application to bioreactor design. In S Furusaki, D Ryu, eds, Advances in Plant Biotechnology: Production of Secondary Metabolites. Elsevier, Amsterdam, The Netherlands, pp 281-305

Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Hilton MG, Walton NJ (1990) Properties of transformed root cultures. In BV Charle- wood, MJC Rhodes, eds, Secondary Metabolites from Plant Tissue Culture. Clarendon Press, Oxford, UK, pp 201-225

Roberts WK, Selitrennikoff CP (1986) Isolation and partial characterization of two antifungal proteins from barley. Biochim Bio- phys Acta 880 161-170

Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. Bioessays 10: 20-24

Saito K, Yamazaki M, Murakoshi I (1992) Transgenic medicinal plants: Agrobncterium-mediated foreign gene transfer and production of secondary metabolites. J Nat Prod 5 5 149-162

Samac DA, Hironaka CM, Yallaly PE, Shah DM (1990) Isolation

1899-1902

262 11628-11633

325-333

86: 896-900

346-356

Dow



ecember 2021


and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thafiana. Plant Physiol93 907-914

Sambrook J, Fritsch EF, Maniatis T (1989) Preparation of monospecific antisera. In Molecular Cloning, A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,

Schlumbaum A, Mauch F, Vogeli U, Boller T (1986) Plant chitinases are potent inhibitors of fungal growth. Nature 324 365-367

Shinshi H, Neuhaus J-M, Ryals j, Meins F (1990) Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol Bioll4 357-368

Singh NK, Bracker CA, Hasagawa PM, Handa AK, Buckel S, Hermodson MA, Pfankoch E, Regnier RE, Bressan RA (1987) Characterization of osmotin. Plant Physiol85 529-536

Sirju-Charran G, Wickham DD (1988) The development of alter- native storage sink sites in sweet potato lpomoea batatas. Ann Bot

Stirpe F, Barbieri L, Battelli MG, Soria M, Lappi DA (1992) Ribosome-inactivating proteins from plants: present status and future prospects. Bio/Technology 1 0 405-412

Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype. Cell 37: 959-967

Toyokawa S, Takeda T, Ogihara Y (1991) Isolation and character-

pp 18.17-18.18

61: 99-102

ization of a new abortifacient protein, karasurin, from root tubers of Trichosanthes kirifowii Max. var. japonicum Kitam. C'hem Pharm

Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178: 362-366

Tsao SW, Yan KT, Yeung HW (1986) Selective killing of choriocar- cinoma cells in vitro by trichosanthin, a plant protein prified from root tubers of the Chinese medicinal herb Trichosanthes kirilowii. Toxicon 24 831-840

Vigers AJ, Roberts WK, Selitrennikoff CP (1991) A new family of plant antifungal proteins. Mol Plant-Microbe Interact 4 315-323

Vogelsang R, Barz W (1993) Purification, characterizaiion and differential hormonal regulation of a B-1,3-glucanase arid two chitinases from chickpea (Cicer arietinum L.). Planta 189 00-69

Wang Y, Qian R-Q, Gu Z-W, Jin S-W, Zhang L-Q, )ria ZX, Tian G-Y, Ni C-Z (1986) Scientific evaluation of Tian Hua Fen (THF)- history, chemistry and application. Pure Appl Chem ! i 8 789-798

Watanabe T, Oyanagi W, Suzuki K, Ohnishi K, Tanaka H (1992) Structure of the gene encoding chitinase D of Bacillus circufans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class 111 plant chitinases. J Bacteriol164 408-414

Yeung H-W, Li WW, Feng Z, Barbieri L, Stirpe F (1588) Trichos- anthin, a-momorcharin, and B-momorcharin: identity of abortifacient and ribosome-inactivating proteins. Int J Pept Protein Res 31:

Bull 39: 716-719

265-268

Dow



ecember 2021

biosynthesis of defense-related proteins in transformed root cultures of trichosanthes kirilowii

Documents