Alteration of flower color in Iris germanica L. 'Fire Bride' through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans.

KEY MESSAGE: Genetic modulation of the carotenogenesis in I. germanica 'Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, 'Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica 'Fire Bride' and produce new flower traits.

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation.

C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar 'Golden Promise'. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed 'Golden Promise'. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of 'Golden Promise' and paralleled the freezing tolerance of the winter hardy barley 'Dicktoo'. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in 'Golden Promise'. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in 'Golden Promise' both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures.

Micropropagation of Iris sp.

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene ß-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

A comparison of the low temperature transcriptomes and CBF regulons of three plant species that differ in freezing tolerance: Solanum commersonii, Solanum tuberosum, and Arabidopsis thaliana.

Solanum commersonii and Solanum tuberosum are closely related plant species that differ in their abilities to cold acclimate; whereas S. commersonii increases in freezing tolerance in response to low temperature, S. tuberosum does not. In Arabidopsis thaliana, cold-regulated genes have been shown to contribute to freezing tolerance, including those that comprise the CBF regulon, genes that are controlled by the CBF transcription factors. The low temperature transcriptomes and CBF regulons of S. commersonii and S. tuberosum were therefore compared to determine whether there might be differences that contribute to their differences in ability to cold acclimate. The results indicated that both plants alter gene expression in response to low temperature to similar degrees with similar kinetics and that both plants have CBF regulons composed of hundreds of genes. However, there were considerable differences in the sets of genes that comprised the low temperature transcriptomes and CBF regulons of the two species. Thus differences in cold regulatory programmes may contribute to the differences in freezing tolerance of these two species. However, 53 groups of putative orthologous genes that are cold-regulated in S. commersonii, S. tuberosum, and A. thaliana were identified. Given that the evolutionary distance between the two Solanum species and A. thaliana is 112-156 million years, it seems likely that these conserved cold-regulated genes-many of which encode transcription factors and proteins of unknown function-have fundamental roles in plant growth and development at low temperature.

Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato.

We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii. Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2 degrees C in S. tuberosum and up to 4 degrees C in S. commersonii. Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBFpathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species.

Glycinebetaine accumulation is more effective in chloroplasts than in the cytosol for protecting transgenic tomato plants against abiotic stress.

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five- to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.

Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield.

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S. tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes (AtCBF1-3) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 degrees C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.

The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato.

The tolerance of various species of plant to abiotic stress has been enhanced by genetic engineering with certain genes. However, the use of such transgenes is often associated with negative effects on growth and productivity under non-stress conditions. The codA gene from Arthrobacter globiformis is of particular interest with respect to the engineering of desirable productive traits in crop plants. The expression of this gene in tomato plants resulted in significantly enlarged flowers and fruits under non-stress conditions. The enlargement of flowers and fruits was associated with high levels of glycinebetaine that accumulated in reproductive organs, such as flower buds and fruits. The enlargement of flowers was related to an increase in the size and number of cells, and reflected the pleiotropic effect of the codA transgene on the expression of genes involved in the regulation of cell division.

Exogenous application of glycinebetaine increases chilling tolerance in tomato plants.

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants are chilling sensitive, and do not naturally accumulate glycinebetaine (GB), a metabolite that functions as a stress protectant. We reported previously that exogenous GB application enhanced chilling tolerance in tomato. To understand its protective role better, we have further evaluated various parameters associated with improved tolerance. Although its effect was most pronounced in younger plants, this benefit was diminished 1 week after GB application. When administered by foliar spray, GB was readily taken up and translocated to various organs, with the highest levels being measured in meristematic tissues, including the shoot apices and flower buds. In leaves, the majority of endogenous GB was found in the cytosol; only 0.6-22.0% of the total leaf GB was localized in chloroplasts. Immediately after GB application, levels of H(2)O(2), catalase activity and expression of the catalase gene (CAT1) were all higher in GB-treated than in control plants. One day after exposure to chilling stress, the treated plants had significantly greater catalase activity and CAT1 expression, although their H(2)O(2) levels remained unchanged. During the following 2 d of this chilling treatment, GB-treated plants maintained lower H(2)O(2) levels but had higher catalase activity than the controls. These results suggest that, in addition to protecting macromolecules and membranes directly, GB-enhanced chilling tolerance may involve the induction of H(2)O(2)-mediated antioxidant mechanisms, e.g. enhanced catalase expression and catalase activity.

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics.

Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwalpha (comprising 29 residues) and Plwbeta (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwalpha, and Plwbeta contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.