Functional Characterization of a New Bifunctional Terpene Synthase LpNES1 from a Medicinal Plant Laggera pter odonta.

Laggera pterodonta, known in China as 'Choulingdan' for its stimulous odor, has long been used as traditional herbal medicine. The essential oil of L. pterodonta, which exhibits various pharmacological activities, is a rich resource of monoterpenes and sesquiterpenes. To date, however, the terpene synthases responsible for their production remain unknown. In present study, a new terpene synthase gene (LpNES1) was identified from L. pterodonta, transcript level of which was significantly upregulated in response to methyl jasmonate treatment. Recombinant LpNES1 could synthesize (E)-nerolidol and minor ß-farnesene from farnesyl diphosphate and linalool from geranyl diphosphate in vitro. Whereas, only sesquiterpenes including (E)-nerolidol and minor ß-farnesene were released when LpNES1 was reconstituted in yeast, even coexpressed with a geranyl diphosphate synthase (ERG20WW). Combined with subcellular localization experiment, the result indicated that the cytosol-targeted LpNES1 was responsible for (E)-nerolidol biosynthesis exclusively in L. pterodonta. Additionally, the expression level of LpNES1 gene was more prominent in floral buds than that in other tissues. LpNES1 characterized in present study not only lays the molecular foundation for sesquiterpene biosynthesis of L. pterodonta, but provides a key element for further biosynthesis of bioactive compound in microbes.

Fine monitoring of major phenolic compounds in lettuce and escarole leaves during storage.

Two varieties of lettuce (Lactuca sativa L. var. crispa and capitata) and one variety of escarole (Cichorium endivia var. latifolium) were chosen due to their different browning sensitivity during storage as minimally processed products. The changes in the compositions and contents of the primary polyphenolic compounds were investigated during the first few hours of storage and then after 1, 2, 3, and 6 days of storage at 6°C and revealed sharp variations. Browning development, activity of phenylalanine ammonia lyase, and concentration of ascorbic acid were also examined during storage. The content of chicoric acid, which was the most abundant phenolic compound, varied substantially during the first 24 hr of storage and between the different varieties. Oak leaf lettuce, which was the most sensitive variety to browning, was characterized by a higher maintained chicoric acid level with a constant decreased chlorogenic acid level during the storage period. PRACTICAL APPLICATIONS: Browning development is responsible for the short shelf life of minimally processed vegetables, such as lettuce (Lactuca sativa). Polyphenolic compounds, as substrates of enzymatic reactions, are involved in the browning susceptibility of leaves. Comparisons of the profiles and contents of these compounds in different leaves showed dramatic variations during storage. This study provides additional information to better control, optimize minimally processed produce and select more suitable leaves for the fresh-cut industry.

Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida.

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only in floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization.

Yeast and mould dynamics in Caciofiore della Sibilla cheese coagulated with an aqueous extract of Carlina acanthifolia All.

Caciofiore della Sibilla is a speciality ewes' milk cheese traditionally manufactured in a foothill area of the Marche region (Central Italy) with a crude extract of fresh young leaves of Carlina acanthifolia All. subsp. acanthifolia as a coagulating agent. The fungal dynamics and diversity of this speciality cheese were investigated throughout the manufacturing and 20-day ripening process, using a combined PCR-DGGE approach. The fungal biota of a control ewes' milk cheese, manufactured with the same batch of milk coagulated with a commercial animal rennet, was also monitored by PCR-DGGE, in order to investigate the contribution of the peculiar vegetable coagulant to the fungal diversity and dynamics of the cheese. Based on the overall results collected, the raw milk and the dairy environment represented the main sources of fungal contamination, with a marginal or null contribution of thistle rennet to the fungal diversity and dynamics of Caciofiore della Sibilla cheese. Copyright © 2016 John Wiley & Sons, Ltd.

Cloning and sequence analysis of the Blumea balsamifera DC farnesyl diphosphate synthase gene.

Blumea balsamifera DC is a member of the Compositae family and is frequently used as traditional Chinese medicine. Blumea balsamifera is rich in monoterpenes, which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viral activities. Farnesyl diphosphate synthase (FPS) is a key enzyme in the biosynthetic pathway of terpenes, playing an important regulatory role in plant growth, such as resistance and secondary metabolism. Based on the conserved oligo amino acid residues of published FPS genes from other higher plant species, a cDNA sequence, designated BbFPS, was isolated from B. balsamifera DC using polymerase chain reaction. The clones were an average of 1.6 kb and contained an open reading frame that predicted a polypeptide of 342 amino acids with 89.07% identity to FPS from other plants. The deduced amino acid sequence was dominated by hydrophobic regions and contained 2 highly conserved DDxxD motifs that are essential for proper functioning of FPS. Phylogenetic analysis indicated that FPS grouped with other composite families. Prediction of secondary structure and subcellular localization suggested that alpha helices made up 70% of the amino acids of the sequence.

The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants.

Triacylglycerol (TAG) is the main storage lipid in plants. Acyl-CoA: diacylglycerol acyltransferase (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferase (PDAT) can catalyze TAG synthesis. It is unclear how these three independent genes are regulated in developing seeds, and particularly if they have specific functions in the high accumulation of unusual fatty acids in seed oil. The expression patterns of DGAT1, DGAT2 and a PDAT in relation to the accumulation of oil and epoxy and hydroxy fatty acids in developing seeds of the plant species Vernonia galamensis, Euphorbia lagascae, Stokesia laevis and castor that accumulate high levels of these fatty acids in comparison with soybean and Arabidopsis were investigated. The expression patterns of DGAT1, DGAT2 and the PDAT are consistent with all three enzymes playing a role in the high epoxy or hydroxy fatty acid accumulation in developing seeds of these plants. PDAT and DGAT2 transcript levels are present at much higher levels in developing seeds of epoxy and hydroxy fatty acid accumulating plants than in soybeans or Arabidopsis. Moreover, PDAT, DGAT1 and DGAT2 are found to be expressed in many different plant tissues, suggesting that these enzymes may have other roles in addition to seed oil accumulation. DGAT1 appears to be a major enzyme for seed oil accumulation at least in Arabidopsis and soybeans. For the epoxy and hydroxy fatty acid accumulating plants, DGAT2 and PDAT also show expression patterns consistent with a role in the selective accumulation of these unusual fatty acids in seed oil.

Effect of glucuronosylation on anthocyanin color stability.

The effect of glucuronosylation on the color stability of anthocyanins was investigated using glucuronosylated anthocyanins isolated from the flower petals of the red daisy (Bellis perennis) or obtained by enzymatic in vitro synthesis using heterologously expressed red daisy glucuronosyltransferase BpUGT94B1. Color stability toward light and heat stress was assessed by monitoring CIELAB color coordinates and stability at pH 7.0 by A(550). Cyanidin-3-O-2''-O-glucuronosylglucoside showed improved color stability in response to light compared to both cyanidin 3-O-glucoside and cyanidin 3-O-2''-O-diglucoside. A similar increase in color stability was not observed following heat treatment. Glucuronosylation did not increase the stability of anthocyanins at pH 7.0 as determined by A(550). To test for a possible effect of glucuronosylation on the color stability of anthocyanins in plant extracts used for food coloration, an elderberry (Sambucus nigra) extract was glucuronosylated in vitro. Glucuronosylation of approximately 50% of total anthocyanins proceeded fast and resulted in increased color stability in response to both heat and light. The data show that glucuronosylation may be used to stabilize industrially used extracts of natural colorants.

Small populations are mate-poor but pollinator-rich in a rare, self-incompatible plant, Hymenoxys herbacea (Asteraceae).

If pollinators or compatible mates are scarce, plants in small populations may be pollen-limited and consequently produce fewer offspring. However, determining the relative importance of the genetic vs ecological mechanisms limiting successful pollination is challenging. We explored the relationships among population size, population connectivity, pollinator visitation, allozyme diversity, mate availability (measured as percent compatible crosses among plants within a population), and pollen limitation in 12 populations (N = 39-885,274) of the self-incompatible plant Hymenoxys herbacea in Ontario, Canada. Unexpectedly, small populations had more insect flower visitors per capitulum than large populations. Consistent with the effects of genetic drift, both allozyme polymorphism and mate availability decreased with decreasing population size. Pollen limitation was low and significant in only one population and could not be predicted based on knowledge of population size, connectivity, compatible mate availability, or pollinator visitation. Population size had detectable effects on both pollinator activity and mate availability. However, because the effect of population size was complementary on these two potentially limiting mechanisms, this threatened plant was rarely pollen-limited.

Isolation and characterization of a novel peroxidase gene ZPO-C whose expression and function are closely associated with lignification during tracheary element differentiation.

In an attempt to elucidate the regulatory mechanism of vessel lignification, we isolated ZPO-C, a novel peroxidase gene of Zinnia elegans that is expressed specifically in differentiating tracheary elements (TEs). The ZPO-C transcript was shown to accumulate transiently at the time of secondary wall thickening of TEs in xylogenic culture of Zinnia cells. In situ hybridization indicated specific accumulation of the ZPO-C transcript in immature vessels in Zinnia seedlings. Immunohistochemical analysis using anti-ZPO-C antibody showed that the ZPO-C protein is abundant in TEs, especially at their secondary walls. For enzymatic characterization of ZPO-C, 6 x His-tagged ZPO-C was produced in tobacco cultured cells and purified. The ZPO-C:6 x His protein had a peroxidase activity preferring sinapyl alcohol as well as coniferyl alcohol as a substrate, with a narrow pH optimum around 5.25. The peroxidase activity required calcium ion and was elevated by increasing Ca2+ concentration in the range of 0-10 mM. An Arabidopsis homolog of ZPO-C, At5g51890, was examined for expression patterns with transgenic plants carrying a yellow fluorescent protein (YFP) gene under the control of the At5g51890 promoter. The YFP fluorescence localization demonstrated vessel-specific expression of At5g51890 in the Arabidopsis roots. Taken collectively, our results strongly suggest that ZPO-C and its homologs play an important role in lignification of secondary cell walls in differentiating TEs.

UDP-glucuronic acid:anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers. Enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis.

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.

Modeling the kinetics of whey protein hydrolysis brought about by enzymes from Cynara cardunculus.

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.

Secoisolariciresinol dehydrogenase purification, cloning, and functional expression. Implications for human health protection.

Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the approximately 32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of approximately 32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated. Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.

Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes.

Arabidopsis thaliana was transformed with a plastidial safflower glycerol-3-phosphate acyltransferase (GPAT) and an Escherichia coli GPAT. The genes were used directly and in modified forms with, as applicable, the plastidial targeting sequence removed, and with an endoplasmic reticulum targeting sequence added. Seeds of plants transformed using only the vector were indistinguishable in oil content from wild-type control plants. All other gene constructs increased seed oil content. The unmodified safflower gene (spgpat) produced oil increases ranging from 10 to 21%. On average, the greatest increase (+22%) was observed in seeds of transformants carrying the spgpat with the targeting peptide removed. The E. coli plsB gene increased seed oil content by an average of 15%.

The molecular cloning of 8-epicedrol synthase from Artemisia annua.

A cDNA library was prepared from Artemisia annua, and a 129-bp fragment was amplified from this library using primers corresponding to sequences conserved in known dicot sesquiterpene synthases. A 1641-bp open reading frame that encoded a predicted protein 35-38% identical to dicot sesquiterpene synthases was cloned using this fragment as a hybridization probe. The gene product expressed in Escherichia coli cyclized farnesyl diphosphate to a 96:4 mixture of (-)8-epicedrol and cedrol. Neither cedrol epimer was detected by GC-MS in an A. annua extract prepared from the same specimen as the cDNA.

Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.

Crosses between sexual and apomictic dandelions (Taraxacum). I. The inheritance of apomixis.

Some dandelions, Taraxacum, are diplosporous gametophytic apomicts. Crosses between closely related diploid sexuals and triploid apomicts were made to study the inheritance of apomixis. Seed-set was less than one-third of that in diploid x diploid crosses, probably because of the inviability of aneuploid pollen or zygotes. Almost 90% of the viable offspring were diploid and the result of selfing, as was shown by a discriminating allozyme marker. Aneuploid outcross pollen had a mentor effect on self-pollen, causing a breakdown of the sporophytic self-incompatibility system. A similar phenomenon has been reported before in wide crosses. Of the 26 allozyme-confirmed hybrids, four were diploids, 15 were triploids and seven were tetraploids. Diploid hybrids were significantly less frequent than triploid hybrids, suggesting either low fitness of haploid pollen or more numerous formation of diploid pollen. Emasculation and bagging of flowers indicated apomictic seed-set in none of the diploid, in one-third of the triploid and in all of the tetraploid hybrids. All apomictic hybrids showed partial seed-set, but additional cross-pollination did not increase seed-set. Cytological analysis of the F2 progeny confirmed that partial apomixis was caused by semisterility and not by residual sexuality (facultative apomixis). The difference in segregation for apomixis between triploid and tetraploid hybrids may be because the triploids originated from partially reduced diploid pollen grains, whereas the tetraploids originated from unreduced triploid pollen grains.

Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

Effects of experimental hypogravity on peroxidase and cell wall constituents in the dwarf marigold.

Dwarf Marigolds grown from seed under experimental hypogravity are modified in lignin content, hemicellulose composition, and peroxidase activity. The two conditions used, clinostats and flotation, induced changes differing in magnitude but qualitatively similar. Most responses on clinostats required corrections for vertical axis rotational effects, thus limiting the value of these instruments in free-fall simulation. These findings extend earlier observations suggesting that increased peroxidase and decreased lignin are characteristic of growth under experimental hypogravity.