Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures.

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.

Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition.

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F(v)/F(m) relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mumol photons m(-2) s(-1) under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mumol photons m(-2) s(-1), under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F(v)/F(m) and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.

Suppression of zeaxanthin formation does not reduce photosynthesis and growth of transgenic tobacco under field conditions.

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with an antisense cDNA construct of violaxanthin de-epoxidase (VDE) was examined for the effects of suppressed xanthophyll-cycle activity on photoinhibition, photosynthesis and growth under field conditions. De-epoxidation of violaxanthin and non-photochemical quenching were highly inhibited in antisense plants relative to vector-control and wild-type plants. However, no differences were observed between antisense and control plants in photosynthetic CO(2) uptake and maximum photochemical yield [(F(m)-F(o))/F(m)] measured at predawn or in actual photochemical yield [(F(m)'-F(s))/F(m)'] measured at midday. Moreover, growth rates of the plants were the same, as were the leaf area ratio, plant height and leaf number. Similarly, antisense plants did not exhibit greater susceptibility to photoinhibition than controls under field conditions. In contrast, when chloroplast protein (D1) synthesis was inhibited by lincomycin, antisense plants were more vulnerable to photoinhibition than wild-type plants. These results indicate that photoprotection under field conditions is not strictly dependent on the levels of the de-epoxidized xanthophylls, antheraxanthin and zeaxanthin.

Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase.

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions.

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 mumol m(-2) s(-1) for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 mumol m(-2) s(-1) under controlled growth conditions as compared to wild-type tobacco.

Developmental expression of violaxanthin de-epoxidase in leaves of tobacco growing under high and low light.

Violaxanthin de-epoxidase (VDE) is a lumen-localized enzyme that catalyzes the de-epoxidation of violaxanthin in the thylakoid membrane upon formation of a transthylakoid pH gradient. We investigated the developmental expression of VDE in leaves of mature tobacco (Nicotiana tabacum) plants grown under high-light conditions (in the field) and low-light conditions (in a growth chamber). The difference in light conditions was evident by the increased pool size (violaxanthin + antheraxanthin + zeaxanthin, VAZ) throughout leaf development in field-grown plants. VDE activity based on chlorophyll or leaf area was low in the youngest leaves, with the levels increasing with increasing leaf age in both high- and low-light-grown plants. However, in high-light-grown plants, the younger leaves in early leaf expansion showed a more rapid increase in VDE activity and maintained higher levels of VDE transcript in more leaves, indicating that high light may induce greater levels of VDE. VDE transcript levels decreased substantially in leaves of mid-leaf expansion, while the levels of enzyme continued to increase, suggesting that the VDE enzyme does not turn over rapidly. The level of VDE changed in an inverse, nonlinear relationship with respect to the VAZ pool, suggesting that enzyme levels could be indirectly regulated by the VAZ pool.

Xanthophyll cycle enzymes are members of the lipocalin family, the first identified from plants.

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the addition and removal of epoxide groups in carotenoids of the xanthophyll cycle in plants. The xanthophyll cycle is implicated in protecting the photosynthetic apparatus from excessive light. Two new sequences for violaxanthin de-epoxidase from tobacco and Arabidopsis are described. Although the mature proteins are well conserved, the transit peptides of these proteins are divergent, in contrast to transit peptides from other proteins targeted to the thylakoid lumen. Sequence analyses of both violaxanthin de-epoxidase and zeaxanthin epoxidase establish the xanthophyll cycle enzymes as members of the lipocalin family of proteins. The lipocalin family is a diverse group of proteins that bind small hydrophobic (lipophilic) molecules and share a conserved tertiary structure of eight beta-strands forming a barrel configuration. This is the first reported identification of lipocalin proteins in plants.

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-L-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.

Characterization of bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase from aspen.

Bispecific O-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremuloides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-L-homocysteine agarose using substrate elution. The enzyme has a M(r) of 40,000 as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both purified and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.

cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen.

A cDNA clone (Ptomt 1) encoding a lignin-bispecific O-methyltransferase (OMT) was isolated by immunological screening of a lambda gt11 expression library prepared from mRNA of developing secondary xylem of aspen (Populus tremuloides). Nucleotide sequence analysis of Ptomt1 revealed an open reading frame of 1095 bp which encodes a polypeptide with a predicted molecular weight of 39,802, corresponding well with the size of the OMT polypeptide estimated by SDS-PAGE. Authenticity of Ptomt1 was demonstrated in part by detection of OMT activity and protein in extracts of Escherichia coli cultures transformed with a plasmid construct containing Ptomt1. In addition, peptides produced from a proteolytic digest of purified OMT and sequenced by automated Edman degradation matched to portions of the deduced amino acid sequence of Ptomt1. Comparison of this sequence to amino acid sequences of OMTs of diverse species identified regions of similarity which probably contribute to the binding site of S-adenosyl-L-methionine. Tissue-specific expression was demonstrated by northern analysis which showed that Ptomt1 hybridized to a 1.7 kb transcript from aspen developing secondary xylem and by tissue printing of aspen stems in which only the outer layer of xylem bound the antibody. A biphasic pattern of gene expression and enzyme activity for OMT was observed from xylem samples of aspen during the growing season which suggests linkage between gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem differentiation in woody plants.

Stress Responses in Alfalfa (Medicago sativa L.): X. Molecular Cloning and Expression of S-Adenosyl-l-Methionine:Caffeic Acid 3-O-Methyltransferase, a Key Enzyme of Lignin Biosynthesis.

S-Adenosyl-l-methionine:caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of caffeic acid to ferulic acid, a key step in the biosynthesis of lignin monomers. We have isolated a functionally active cDNA clone (pCOMT1) encoding alfalfa (Medicago sativa L.) COMT by immunoscreening a lambdaZAPII cDNA expression library with anti-(aspen COMT) antibodies. The derived amino acid sequence of pCOMT1 is 86% identical to that of COMT from aspen. Southern blot analysis indicates that COMT in alfalfa is encoded by at least two genes. Addition of an elicitor preparation from bakers' yeast to alfalfa cell suspension cultures resulted in a rapid accumulation of COMT transcripts, which reached a maximum level around 19 hours postelicitation. Northern blot analysis of total RNA from different organs of alfalfa plants at various developmental stages showed that COMT transcripts are most abundant in roots and stems. Transcripts encoding ATP: i-methionine-S-adenosyl transferase (AdoMet synthetase, EC 2.5.1.6), the enzyme responsible for the synthesis of the methyl donor for the COMT reaction, were coinduced with COMT transcripts in elicitor-treated cells and exhibited a similar pattern of expression to that of COMT in different organs of alfalfa plants at various stages of development.

Phenolic Compound Utilization by the Soft Rot Fungus Lecythophora hoffmannii.

Nine phenolic compounds were metabolized by the soft rot fungus Lecythophora hoffmannii via protocatechuic acid and subsequently cleaved by protocatechuate 3,4-dioxygenase as determined by oxygen uptake, substrate depletion, and ring cleavage analysis. Catechol was metabolized by catechol 1,2-dioxygenase. Fungal utilization of these aromatic compounds may be important in the metabolism of wood decay products.