Cutinsomes and cuticle enzymes GPAT6 and DGAT2 seem to travel together from a lipotubuloid metabolon (LM) to extracellular matrix of O. umbellatum ovary epidermis.

In the ovary epidermis of O. umbellatum there are lipotubuloid metabolons (LMs), in which synthesis of lipids takes place. This process partly provides nourishment, and partly cuticle building blocks, transformed, among others, with the participation of cutinsomes. The cutinsomes are cutin-building structures, 40-200nm in size, which are formed as a result of self-assembly and self-esterification of hydroxy fatty acids. The cutinsomes, by binding to the cuticle, introduce into it nonlinear, amorphous and cross-linked polymers. Double-immunogold EM observations revealed that enzymes producing elements of cutin (GPAT6) and waxes (WS/DGAT) were found not only as free cytoplasmic molecules but also in many cases they were bound to carboxylate-carboxylic shell of cuntinsomes. Hence, we suppose that these enzymes can move alone or together with the cutinsomes through cytoplasm (pH 6.8-7.0), plasmalemma and the polysaccharide layer of a cell wall to the site of their functioning i.e. to the cuticle (pH 5.0).

[Physiological and biochemical change of Paris seed in after-ripening during variable temperature stratification].

In order to explore the dormancy physiological and biochemical mechanism of Paris seeds, the seed embryo growth courses, and the dynamic change of 5 enzymes, include SOD, POD, CAT, MDH, G-6-PDH were measured during variable temperature stratification. The results indicated that Paris seeds embryo grew quickly after 40 d in warm-stratification (18 ± 1) °C, at the meantime the metabolic activity was significantly strengthened. These facts showed that Paris seeds turned into physiological after-ripening process. After 60-80 d, the morphological embryo after-ripping process basically completed, and the following cold-stratification (4 ± 1) °C furthered Paris seed to finish physiological after-ripening. After 40 d, the activity of MDH decreased while G-6-PDH increased significantly. This showed that the main respiratory pathway of seed changed from TCA to PPP, which benifited breaking seed dormancy. In the whole period of stratification process, the activity variation of SOD and CAT was insignificantly and the activity of POD was enhanced significantly after shifting the seed in cold stratification process. This showed that SOD, CAT had no direct effects on breaking Paris seed dormancy but keeping the seed vigor, while the POD might involve in the process of Paris seed dormancy breaking.

Metabolic fate of cardiac glycosides and flavonoids upon fermentation of aqueous sea squill (Drimia maritima L.) extracts.

Sea squill (Drimia maritima L.) extracts have been used for centuries for the medical treatment of heart diseases. A procedure for the preparation of Drimia extracts applied for such purposes comprising a fermentation step is described in the German Homoeopathic Pharmacopoeia (GHP). However, little is known about the secondary metabolite profile of such extracts and the fate of these components upon processing and storage. Thus, in the present study sea squill extracts were monitored during fermentation and storage by HPLC-DAD-MS(n) and GC-MS to characterise and quantitate individual cardiac glycosides and phenolic compounds. For this purpose, a previously established HPLC method for the separation and quantitation of pharmacologically relevant cardiac glycosides (bufadienolides) was validated. Within 12 months of storage, total bufadienolide contents decreased by about 50%, which was attributed to microbial and plant enzyme activities. The metabolisation and degradation rates of individual bufadienolide glycosides significantly differed, which was attributed to differing structures of the aglycones. Further degradation of bufadienolide aglycones was also observed. Besides reactions well known from human metabolism studies, dehydration of individual compounds was monitored. Quantitatively predominating flavonoids were also metabolised throughout the fermentation process. The present study provides valuable information about the profile and stability of individual cardiac glycosides and phenolic compounds in fermented Drimia extracts prepared for medical applications, and expands the knowledge of cardiac glycoside conversion upon microbial fermentation.

Cloning and characterization of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene from Paris fargesii Franch.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) plays an important role in catalyzing the first committed step of isoprenoids biosynthesis in mevalonic acid (MVA) pathway. Here, we cloned a full-length transcript of Paris fargesii Franch. The full-length cDNA of P. fargesii HMGR (Pf-HMGR, GenBank accession no. JX508638) was 1,973 bp and contained a 1,728 bp ORF encoding 576 amino acids. Sequence analysis revealed that the deduced Pf-HMGR had high similarity with HMGRs from other plants, including Ricinus communis (77%), Litchi chinensis (76%), Michelia chapensis (75%) and Panax quinquefolius (72%). It had a calculated molecular mass of about 62.13 kDa and an isoelectric point (pI) of 8.47. It contained two transmembrane domains, two putative HMGR binding sites and two NADP(H)-binding sites. The predicted 3-D structure revealed that Pf-HMGR had a similar spatial structure with other plant HMGRs. Three catalytic regions, including L-domain, N-domain and S-domain were detected by structural modeling of HMGR. Tissue expression analysis revealed that Pf-HMGR was strongly expressed in roots and stems than in leaves. Taken together, our data laid a foundation for further investigation of HMGR's functions and regulatory mechanisms in plants.

Molecular cloning and characterization of a novel adenylyl cyclase gene, HpAC1, involved in stress signaling in Hippeastrum x hybridum.

Adenylyl cyclases (ACs) are enzymes that generate cyclic AMP, which is involved in different physiological and developmental processes in a number of organisms. Here, we report the cloning and characterization of a new plant adenylyl cyclases (AC) gene, designated HpAC1, from Hippeastrum x hybridum. This gene encodes a protein of 206 amino acids with a calculated molecular mass of 23 kD and an isoelectric point of 5.07. The predicted amino acid sequence contains all the typical features of and shows high identity with putative plant ACs. The purified, recombinant HpAC1 is able to convert ATP to cAMP. The complementation test that was performed to analyze the ability of HpAC1 to compensate for the AC deficiency in the Escherichia coli SP850 strain revealed that HpAC1 functions as an adenylyl cyclase and produces cyclic AMP. Moreover, it was shown that the transcript level of HpAC1 and cyclic AMP concentration changed during certain stress conditions. Both mechanical damage and Phoma narcissi infection lead to two sharp increases in HpAC1 mRNA levels during a 72-h test cycle. Changes in intracellular cAMP level were also observed. These results may indicate the participation of a cAMP-dependent pathway both in rapid and systemic reactions induced after disruption of symplast and apoplast continuity.

[Molecular cloning of squalene synthase gene form Paris polyphylla and its expression in Escherichia coli].

OBJECTIVE: To clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS. METHOD: Using homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS. RESULT: The full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG. CONCLUSION: The full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.

Dissecting the stress metabolic alterations in in vitro Cyrtanthus regenerants.

Metabolic plasticity in plants allows for continuous adjustments of defence strategies in suboptimal environments. Proline and other metabolites figure prominently in most stress-mediated responses. This study examined the expression of salinity and osmotic adjustments in the enzymatic activity and accumulation of solutes and metabolites in response to imposed water and salt stress in Cyrtanthus contractus N.E. Br. and Cyrtanthus guthrieae L. Bolus regenerants. In vitro-derived plantlets were cultured on solid Murashige and Skoog (MS) media with three different polyethylene glycol (PEG)-induced osmotic levels and four NaCl stress-induced levels at 25 °C. The levels of proline and phenolic compounds measured at intervals of three, four and five weeks from initial plantlet culture increased in a stress-dependent pattern. The levels of these metabolites also showed a significant increase with an increase in the duration of plantlets under stress conditions. The highest proline concentration (9.98 µmol g(-1) FW) was recorded in C. contractus at 300 µM NaCl after five weeks. A corresponding high level of total phenolic compounds (147 mg GAE g(-1) DW) was also recorded in the same treatment for the same species. The activity of proline dehydrogenase (PDH) (EC 1.5.99.8) was shown to decrease with an increase in proline levels from week three to week five in almost all the stress conditions. The high levels, particularly of phenolic compounds obtained under osmotic and salinity stress conditions in this study present a promising potential of manipulating culture and/or growing conditions for improved secondary compound production and hence medicinal benefits.

Characterization of Squalene synthase gene from Chlorophytum borivilianum (Sant. and Fernand.).

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.

Alkaloids from Chlidanthus fragrans and their acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase activities.

Eleven Amaryllidaceae alkaloids (1-11) were isolated from fresh bulbs of Chlidanthus fragrans Herb. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic experiments. Complete NMR assignments were achieved for deoxypretazzetine (1). All compounds were evaluated for their erythrocytic acetylcholinesterase and serum butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. In biological assays, only the crinine type Amaryllidaceae alkaloid undulatine showed promising acetylcholinesterase and prolyl oligopeptidase inhibition activity with IC50 values of 23.0 +/- 1.0 microM and 1.96 +/- 0.12 mM, respectively. Other isolated compounds were considered inactive.

Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus.

A cDNA encoding an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AaAA7GT) was isolated from Agapanthus africanus petals; this is the first AAGT identified in a monocot. Peak expression of AaAA7GT in developing A. africanus petals occurred before the flowering stage, and was later than found previously for other anthocyanin biosynthetic genes. Analysis of recombinant proteins showed AaAA7GT had strict substrate preference for anthocyanidin 3-O-glycosides. The AaAA7GT amino acid had high sequence similarity to glycoside hydrolase family 1 (GH1) proteins, which typically act as ß-glycosidases. A phylogenetic analysis of amino acid sequences suggested that AAGTs were derived from glycosidase early in the angiosperm lineage.

Cryo-tolerance of zygotic embryos from recalcitrant seeds in relation to oxidative stress--a case study on two amaryllid species.

Oxidative stress is a major component of cryoinjury in plant tissues. This study investigated the ability of recalcitrant (i.e. desiccation sensitive) Amaryllis belladonna L. and Haemanthus montanus Baker zygotic embryos to survive cryopreservation, in relation to oxidative stress. The study also investigated whether glycerol cryoprotection promoted embryo post-cryo survival by protecting enzymic antioxidant activities. Zygotic embryos excised from hydrated stored seeds were subjected to various combinations of rapid dehydration (to < or >0.4 g g⁻¹ [dmb]), cryoprotection (with sucrose or glycerol), and cooling (either rapidly or slowly), and were thereafter assessed for viability, extracellular superoxide (·O2⁻) production, lipid peroxidation (TBARS) and antioxidant enzyme activities. Short-term hydrated storage of whole seeds was accompanied by ·O2⁻ production and lipid peroxidation, but ·O2⁻ levels were lower than in dehydrated and cooled embryos and viability was 100%, possibly associated with the high activities of certain antioxidant enzymes. Partial dehydration and cryoprotection (in H. montanus only) increased ·O2⁻ production (especially in cryoprotected-dried embryos) and was associated with some viability loss, but this was not correlated with enhanced lipid peroxidation. Cooling was generally accompanied by the greatest increase in ·O2⁻ production, and with a decline in viability. In A. belladonna only, post-cryo TBARS levels were generally higher than for fresh and pre-conditioned embryos. Partial dehydration and cooling decreased antioxidant activities, but these were consistently less severe in glycerol cryoprotected-dried, as opposed to non-cryoprotected-dried embryos. Post-cryo viability retention for glycerol cryoprotected-dried embryos was significantly higher than for non-cryoprotected-dried embryos, possibly facilitated by relatively low post-drying TBARS levels and high post-drying and post-rewarming activities of some antioxidant enzymes in the former. Pre-conditioning treatments such as glycerol cryoprotection, when used in combination with partial drying, may enhance post-cryo viability retention in recalcitrant zygotic embryos by protecting the activities of certain antioxidant enzymes during pre-conditioning for, and after retrieval from, cryostorage.

Alkaloid patterns in Leucojum aestivum shoot culture cultivated at temporary immersion conditions.

The alkaloid patterns in Leucojum aestivum L. shoot culture cultivated at temporary immersion conditions were investigated using gas chromatography-mass spectrometry. 18 alkaloids were identified, and galanthamine, hamayne and lycorine were dominant. The L. aestivum 80 shoot culture, cultivated at temporary immersion conditions, is a prospective biological matrix for obtaining wide range Amaryllidaceae alkaloids, showing valuable biological and pharmacological activities. The temperature of cultivation influenced enzyme activities, catalyzing phenol oxidative coupling of 4'-O-methylnorbelladine and formation of the different groups Amaryllidaceae alkaloids. Decreasing the temperature of cultivation of L. aestivum 80 shoot culture led to activation of para-ortho' phenol oxidative coupling (formation of galanthamine type alkaloids) and inhibited ortho-para' and para-para' phenol oxidative coupling (formation of lycorine and haemanthamine types alkaloids).

First insights into the mode of action of a "lachrymatory factor synthase"--implications for the mechanism of lachrymator formation in Petiveria alliacea, Allium cepa and Nectaroscordum species.

A study of an enzyme that reacts with the sulfenic acid produced by the alliinase in Petiveria alliacea L. (Phytolaccaceae) to yield the P. alliacea lachrymator (phenylmethanethial S-oxide) showed the protein to be a dehydrogenase. It functions by abstracting hydride from sulfenic acids of appropriate structure to form their corresponding sulfines. Successful hydride abstraction is dependent upon the presence of a benzyl group on the sulfur to stabilize the intermediate formed on abstraction of hydride. This dehydrogenase activity contrasts with that of the lachrymatory factor synthase (LFS) found in onion, which catalyzes the rearrangement of 1-propenesulfenic acid to (Z)-propanethial S-oxide, the onion lachrymator. Based on the type of reaction it catalyzes, the onion LFS should be classified as an isomerase and would be called a "sulfenic acid isomerase", whereas the P. alliacea LFS would be termed a "sulfenic acid dehydrogenase".

Modulation of inhibitory activity of xylanase-α-amylase inhibitor protein (XAIP): binding studies and crystal structure determination of XAIP-II from Scadoxus multiflorus at 1.2 Å resolution.

BACKGROUND: Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature. RESULTS: In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR). CONCLUSION: The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-ß3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop ß6-α6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10(-6) M and 3.4 × 10(-8) M for the dissociation constants of XAIP-II with xylanase GH11 and α-amylase GH13 respectively and those of 4.5 × 10(-7) M and 3.6 × 10(-6) M of XAIP with xylanase GH11 and α-amylase GH13 respectively.

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.

An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes.

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.

Chromosome termini of the monocot plant Othocallis siberica are maintained by telomerase, which specifically synthesises vertebrate-type telomere sequences.

Lack of Arabidopsis-type T3AG3 telomere sequences has recently been reported for the majority of investigated taxa of the monocot order Asparagales. In order to investigate this phenomenon in more detail, we conducted extensive cytogenetic and molecular analyses of the telomeres in Othocallis siberica, a member of this order. Terminal restriction fragment analysis together with Bal31 exonuclease assay showed that chromosome termini in O. siberica are formed by long stretches (more than 10 kbp) of vertebrate-type T2AG3 repeats. In addition, telomerase activity specifically synthesising (T2AG3)n sequence was detected in O. siberica protein extracts by telomerase repeat amplification protocol (TRAP). Fluorescence in situ hybridisation (FISH) revealed the presence of the vertebrate-type T2AG3 telomere sequences at all chromosome termini and at a few additional regions of O. siberica chromosomes, whereas Arabidopsis-type T3AG3 DNA and peptide nucleic acid (PNA) probes did not hybridise to chromosomes of Othocallis, except for polymorphic blocks in chromosomes 2 (interstitial) and 4 (terminal). These interstitial/terminal regions are apparently composed of large blocks of (T2AG3)n and (T3AG3)n DNA and represent a unique example of interspersion of two types of telomeric repeats within one genome. This may be a reflection of the recent evolutionary switch from Arabidopsis- to vertebrate-type telomeric repeats in this plant group.

Telomere variability in the monocotyledonous plant order Asparagales.

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the 'typical' Arabidopsis-type telomere (TTTAGGG)(n). This stimulated us to determine what has replaced these sequences. Using slot-blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis-type telomeric sequence has been partly or fully replaced by the human-type telomeric sequence (TTAGGG)(n). Species in Allium lack the human-type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis, Bombyx (TTAGG)(n) and Tetrahymena (TTGGGG)(n). FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human-type motif. These motifs probably form the 'true telomere' but the error rate of motif synthesis is higher compared with 'typical' plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis-like telomeres to a low-fidelity synthesis of human-like telomeres.

Phylogeny, concerted convergence, and phylogenetic niche conservatism in the core Liliales: insights from rbcL and ndhF sequence data.

Calochortus and the family Liliaceae s.s. have often been considered each other's closest relatives, based partly on their shared possession of bulbs, visually showy flowers, winged wind-dispersed seeds, and narrow parallel-veined leaves. We present a well-supported molecular phylogeny for these groups and their close relatives in the core Liliales, based on sequence variation in the chloroplast-encoded rbcL and ndhF genes. This analysis identifies Liliaceae s.s. as monophyletic. including one clade (((Lilium, Fritillaris, Nomocharis), Cardiocrinum), Notholirion) that appears to have diversified in the Himalayas roughly 12 million years ago and another ((Erythronium, Tulipa), (Gagea, Lloydia)) that arose in East Asia at about the same time. Medeola and Clintonia are sister to Liliaceae s.s. and bear rhizomes, inconspicuous flowers, fleshy animal-dispersed fruits, and broad reticulate-veined leaves. Calochortus is sister to Tricyrtis; both Tricyrtis and the neighboring clade of Prosartes-Streptopus-Scoliopus share several of the traits seen in Medeola-Clintonia. The core Liliales thus provide compelling examples of both concerted convergence and phylogenetic niche conservatism. Invasion of open, seasonal habitats was accompanied by the independent evolution of bulbs, showy flowers, wind-dispersed seeds, and narrow parallel-veined leaves in Calochortus and Liliaceae s.s. Conversely, persistence in shady habitats was accompanied by the retention of rhizomes, inconspicuous flowers, animal-dispersed seeds, and broad reticulate-veined leaves in their sister groups. We advance arguments for the context-specific adaptive value of each of these traits, as well as evidence of parallel trends in other groups. Concerted convergence--convergence in several different traits, favored by the same shared set of ecological conditions, in two or more lineages--is an important evolutionary process that can mislead evolutionary analyses based solely on phenotypic variation.

Organization of cell walls in Sandersonia aurantiaca floral tissue.

Visual symptoms of the onset of senescence in Sandersonia aurantiaca flowers begin with fading of flower colour and wilting of the tissue. When fully senescent, the flowers form a papery shell that remains attached to the plant. The cell walls of these flowers have been examined to determine whether there are wall modifications associated with the late stages of expansion and subsequent senescence-related wilting. Changes in the average molecular size of pectin were limited through flower opening and senescence, although there was a loss of neutral sugar-containing side-branches from pectins in opening flowers, and the total amounts of pectin and cellulose continued to rise in cell walls of fully senescent sandersonia flowers. Xyloglucan endotransglycosylase activity increased in opening and mature flowers, but declined sharply as flowers wilted. Concomitantly, the proportion of hemicellulose polymers of increasing molecular weight increased from flower expansion up to the point at which wilting occurred. Approximately 50% of the non-cellulosic neutral sugar in mature flower cell walls was galactose, primarily located in an insoluble cell wall fraction. Total galactose in this fraction increased per flower with maturity, then declined at the onset of wilting. Beta-galactosidase activity was low in expanding tepals, but increased as flowers matured and wilted.