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 μ mol 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 μ mol 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.

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.

Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.

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 μmol 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 μmol m −2 s −1 under controlled growth conditions as compared to wild-type tobacco.

SummaryThe xanthophyll cycle is the cyclical interconversion of violaxanthin, antheraxanthin and zeaxanthin in plants and green algae. The existence of the cycle has been known for many years but has attracted renewed interest because of its role in protection of plants against the potentially harmful effects of excess light by enhancing the dissipation of excess energy as heat. The cycle is catalyzed by two enzymes that are localized on opposite sides of the thylakoid membrane. The de-epoxidase that converts violaxanthin to zeaxanthin by way of the intermediate, antheraxanthin, is localized in the thylakoid lumen. The epoxidase that catalyzes the resynthesis of violaxanthin is bound to the stromal side of the membrane. The extent and rate of zeaxanthin and antheraxanthin formation (de-epoxidation) are affected by at least four factors, namely, (i) pool size, (ii) availability, (iii) ascorbate, and (iv) lumen pH. The mechanism for de-epoxidation is assumed to be reduction followed by dehydration. Factors affecting the recovery of violaxanthin (epoxidation) include levels of NADPH, ferredoxin, ferredoxin-oxidoreductase and FAD. The mechanism of epoxidation is assumed to be similar to other monooxygenases wherein hydroperoxyflavin is involved and one oxygen atom from molecular oxygen is incorporated. Recently, the cDNAs for both enzymes were isolated and catalytic activities of the expressed proteins demonstrated. Analyses of the deduced polypeptide sequences indicate that both proteins belong to the lipocalin family. The lipocalins are a diverse group of proteins with a conserved barrel structure that bind small hydrophobic molecules. This chapter summarizes the biochemistry of the xanthophyll cycle and examines recent advances in the molecular biology of the cycle.

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.

A sugarcane (Saccharum spp.) leaf cDNA library was screened with a 1.4-kb EcoRV-BsmI fragment of pTF414A (Glc transporter cDNA from Arabidopsis thaliana), which includes most of the coding region of the cDNA (Sauer et al., 1990). Plaque hybridizations were performed at low strin-gency with the random primed 32P-labeled fragment of pTF414A followed by exposure of the nitrocellulose mem-branes to film for 48 h at -7OOC. The cDNA of one of the positive clones was subcloned and sequencing revealed a cDNA of 1279 nucleotides. This cDNA (SMPl) contained an open reading frame encoding a polypeptide of 325 amino acids with a predicted molecular mass of 35.8 kD (Table I). The nucleotide sequence around the first Met of this open reading frame has 67% homology to the consensus translation initiation site of plants (Joshi, 1987). Hydropathy analysis indicated that the SMPl protein is a putative membrane protein. The grand average hydropathy value of the SMPl protein was 0.38, significantly greater than the mean for soluble proteins (-0.4) (Kyte and Doolittle, 1982). This value is similar to values for membrane transport proteins for Glc (0.48) (Sauer et al., 1990) and SUC (0.57) (Riesmeier et al., 1992). In addition, SMPl protein has a calculated isoelectric point (8.62) that is similar to that of the membrane transport proteins for Glc (9.49) and SUC (8.81). a hydropathy plot revealed that the deduced protein six putative membrane-spanning regions of 19 to 26 amino acids, an average hydropathy index of >1.6

Apoplastic movement of carbohydrates from source to sink tissues is mediated by membrane transport proteins. Isolating cDNA clones for Glc and Suc transport proteins and using these cDNAs to alter expression of the transporters will help to elucidate their role in carbon allocation. The first cDNA for a carbohydrate transporter from a higher plant was recently cloned from Arabidopsis thaliana (Sauer et al., 1990). This cDNA encodes a Glc transporter as determined by heterologous expression in the yeast Schizosaccharomyces pombe. We have isolated similar cDNAs from sugarcane (Saccharum spp.) using this cDNA as a hybridization probe. A sugarcane mature leaf cDNA library was screened at low stringency with the coding region (1.4-kb EcoRV-BsmI fragment of pTF414A) of the A. thaliana Glc transporter (Sauer et al., 1990). The insert of one of the positive clones (SGT1) was subcloned, and sequencing revealed a 1215-bp cDNA. SGTl is a partial cDNA containing an open reading frame encoding a polypeptide of 287 amino acids. The deduced polypeptide shares 56.4% identity and 72.5% similarity to the A. thaliana Glc transporter deduced protein. The leaf library was rescreened at high stringency using SGTl as a probe. Ten positive X clones were plaque purified, one of which had an insert large enough for a complete open reading frame. This 1726-bp cDNA (SGT2) contained an open reading frame encodmg a polypeptide of 518 amino acids with a calculated molecular m a s of 55.7 kD. The translation initiation sequence at the first Met of this open reading frame is identical in seven of nine nucleotides to the consensus sequence for plants (Lutcke et al., 1987). A stop codon in the same reading frame is present 63 bp upstream of this AUG codon. The coding regions of the two cDNAs are identical except for a single nucleotide base change that results in a conservative amino acid difference of an Ala in SGTl and a Val in SGT2. However, the 3‘ untranslated regions differ greatly in

A cDNA clone (Ptomt1) 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 39802, 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.

S-Adenosyl-L-methionine:caffeic acid3-0-methyltransferase (COMT,EC2.1.1.6) catalyzes theconversion ofcaffeic acidto ferulic acid, akeystepinthebiosynthesis oflignin monomers. We haveisolated a functionally active cDNAclone(pCOMT1) encoding alfalfa (Medicago sativa L.) COMTbyimmunoscreening a XZAPII cDNAexpression library withanti-(aspen COMT)anti- bodies. Thederived aminoacidsequenceofpCOMT1is86% identical tothatofCOMT fromaspen.Southern blotanalysis indicates thatCOMTinalfalfa isencodedbyatleast twogenes. Addition ofanelicitor preparation frombakers' yeasttoalfalfa cell suspension cultures resulted inarapid accumulation ofCOMT transcripts, whichreached a maximumlevel around19hours postelicitation. Northern blot analysis oftotal RNAfromdifferent organs ofalfalfa plants atvarious developmental stages showed thatCOMTtranscripts aremostabundant inrootsandstems. Transcripts encoding ATP:i-methionine-S-adenosyl transferase (AdoMet synthetase, EC2.5.1.6), theenzymeresponsible forthe synthesis ofthemethyl donorfortheCOMTreaction, wereco- induced withCOMTtranscripts inelicitor-treated cells andexhib- ited asimilar pattern ofexpression tothatofCOMTindifferent organs ofalfalfa plants atvarious stages ofdevelopment.