Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997     

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective medium containing 200 μg ml⁻¹ kanamycin and 500 μg ml⁻¹ carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode. Received: 13 July 1999 / Accepted: 8 August 1999     

The management of potato cyst nematodes using resistant Solanaceae potato clones as trap crops

The concept of using a range of Solanaceae potato clones as trap crops for potato cyst nematode (PCN) management was investigated. A series of field trials were undertaken from 1999 to 2002 that evaluated 10 clones of either wild Solanum potato species, breeder’s hybrid lines or commercial cultivars. All had high resistance to all known PCN pathotypes (both Globodera rostochiensis and Globodera pallida) and the ability to stimulate high levels of PCN hatch. Investigations showed potential for the development of some clones as a means of reducing high PCN field population levels and for use by organic potato producers.     

TMV resistance gene N homologues are linked to Synchytrium endobioticum resistance in potato

 The fungus Synchytrium endobioticum, the causal agent of potato wart disease, is subject to world-wide quarKantine regulations due to the production of persistent resting spores and lack of effective chemical control measures. The selection of Synchytrium-resistant potato cultivars may be facilitated by using markers closely linked with a resistance gene or by transferring a cloned gene for resistance into susceptible cultivars. Sen1, a gene for resistance to Synchytrium endobioticum race 1, was localized on potato chromosome XI in a genomic region which is related to the tobacco genome segment harbouring the N gene for resistance to TMV. Using N as probe, we isolated homologous cDNA clones from a Synchytrium-resistant potato line. The N-homologous sequences of potato identified by RFLP mapping a family of resistance gene-like sequences closely linked with the Sen1 locus. Sequence analysis of two full-length N-homologous cDNA clones revealed the presence of structural domains associated with resistance gene function. One clone (Nl-25) encodes a polypeptide of 61 kDa and harbours a Toll-interleukin like region (TIR) and a putative nucleotide binding site (NBS). The other clone (Nl-27) encodes a polypeptide of 95 kDa and harbours besides the TIR and NBS domains five imperfect leucine-rich repeats (LRRs). Both clones have at their amino terminus a conserved stretch of serine residues that was also found in the N gene, the RPP5 gene from Arabidopsis thaliana and several other resistance gene homologues, suggesting a function in the resistance response. Cloning of the disease resistance locus based on map position and the establishment of PCR-based marker assays to assist selection of wart resistant potato genotypes are discussed. Received: 4 August 1998 / Accepted: 14 August 1998     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999     

Resistance to late blight in Solanum bulbocastanum is mapped to chromosome 8

Somatic hybrids between potato and Solanum bulbocastanum, a wild diploid (2n=2x=24) Mexican species, are highly resistant to late blight, caused by Phytophthora infestans. Both randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers that are closely linked to the resistance have been noted by analysis of three different backcross-2 populations derived from two different somatic hybrids. With reference to previously published potato and tomato maps, resistance appears to be on the long arm of chromosome 8 and is flanked by RFLP markers CP53 and CT64. In a population of BC₂ plants derived from a cross between the BC₁ line J10lK6 [(S. tuberosum PI 203900+S. bulbocastanum PI 243510) ×Katahdin)]×Atlantic, late blight resistance cosegregated with RFLP marker CT88 and RAPD marker OPG02–625. Received: 26 November 1999 / Accepted: 22 December 1999     

Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

A cDNA encoding a novel inwardly rectifying potassium (K⁺ _in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K⁺ _in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K⁺-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺ _in channels. The pharmacological inhibitor Cs⁺, applied externally, inhibited K⁺ _in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC₅₀) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K⁺ into root hair cells. Comparison of homologous K⁺ _in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships. Received: 3 August 1999 / Accepted: 2 November 1999     

Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda

 Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When ¹⁵NO₃ ⁻ pulse-chase experiments with intact and induced plants were used to follow the incorporation of ¹⁵N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of ¹⁵N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores. Received: 3 July 1999 / Accepted: 17 September 1999     

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata

The concentration of cytoplasmic free calcium ([Ca²⁺]_cyt) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca²⁺]_cyt were applied by cold treatment, and ion injection was also used to increase [Ca²⁺]_cyt, by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca²⁺]_cyt was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca²⁺]_cyt (from 107 to 210 nM). In response to iontophoresis of Ca²⁺, plasmodesmata were observed to go from “open” (low resistance) to “shut” (high resistance) and then back “open” within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca²⁺]_cyt. Received: 2 June 1999 / Accepted: 16 July 1999     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator

 The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated from the C₄ plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C₃ plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more ¹⁴CO₂ in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the rate of CO₂ assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. Received: 26 March 1999 / Accepted: 21 August 1999     

Turing instabilities in general systems

We present necessary and sufficient conditions on the stability matrix of a general n(≥2)-dimensional reaction-diffusion system which guarantee that its uniform steady state can undergo a Turing bifurcation. The necessary (kinetic) condition, requiring that the system be composed of an unstable (or activator) and a stable (or inhibitor) subsystem, and the sufficient condition of sufficiently rapid inhibitor diffusion relative to the activator subsystem are established in three theorems which form the core of our results. Given the possibility that the unstable (activator) subsystem involves several species (dimensions), we present a classification of the analytically deduced Turing bifurcations into p (1 ≤p≤ (n− 1)) different classes. For n = 3 dimensions we illustrate numerically that two types of steady Turing pattern arise in one spatial dimension in a generic reaction-diffusion system. The results confirm the validity of an earlier conjecture [12] and they also characterise the class of so-called strongly stable matrices for which only necessary conditions have been known before [23, 24]. One of the main consequences of the present work is that biological morphogens, which have so far been expected to be single chemical species [1–9], may instead be composed of two or more interacting species forming an unstable subsystem. Received: 21 September 1999 / Revised version: 21 June 2000 / Published online: 24 November 2000     

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism

 The negative gravitropism of the sporangiophores of Phycomyces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132–142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15–25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains. Received: 2 September 1999 / Accepted: 9 November 1999     

Tolerance and overcompensation to infection by Phytophthora infestans in the wild perennial climber Solanum dulcamara

Studies of infection by Phytophthora infestans—the causal agent of potato late blight—in wild species can provide novel insights into plant defense responses, and indicate how wild plants might be influenced by recurrent epidemics in agricultural fields. In the present study, our aim was to investigate if different clones of Solanum dulcamara (a relative of potato) collected in the wild differ in resistance and tolerance to infection by a common European isolate of P. infestans. We performed infection experiments with six S. dulcamara genotypes (clones) both in the laboratory and in the field and measured the degree of infection and plant performance traits. In the laboratory, the six evaluated genotypes varied from resistant to susceptible, as measured by degree of infection 20 days post infection. Two of the four genotypes susceptible to infection showed a quadratic (concave downward) relationship between the degree of infection and shoot length, with maximum shoot length at intermediate values of infection. This result suggests overcompensation, that is, an increase in growth in infected individuals. The number of leaves decreased with increasing degree of infection, but at different rates in the four susceptible genotypes, indicating genetic variation for tolerance. In the field, the inoculated genotypes did not show any disease symptoms, but plant biomass at the end of the growing season was higher for inoculated plants than for controls, in‐line with the overcompensation detected in the laboratory. We conclude that in S. dulcamara there are indications of genetic variation for both resistance and tolerance to P. infestans infection. Moreover, some genotypes displayed overcompensation. Learning about plant tolerance and overcompensation to infection by pathogens can help broaden our understanding of plant defense in natural populations and help develop more sustainable plant protection strategies for economically important crop diseases.     

Autonomous cell death, temperature sensitivity and the genetic control associated with resistance to cucumber mosaic virus (CMV) in diploid potatoes (Solanum spp.)

Cucumber mosaic virus (CMV) is a commonly occurring plant virus that causes severe damage in many crops, including the diploid crop species tomato and pepper (Lycopersicon spp. and Capsicum spp., respectively) of the family Solanaceae, but it is neither common nor economically important in cultivated potatoes (Solanum tuberosum; Solanaceae). Resistance to CMV was examined in two diploid (2n=2x=24), highly heterozygous potato populations (Solanum spp.; Solanaceae) consisting of 76 and 126 progeny. Resistance to long-distance transport of CMV controlled by one locus with a major effect and functional at a low temperature (18°C) but overcome at a high temperature (28°C) was identified in one population. In the other population, resistance was controlled by two loci with major effects. In both populations, additional genes with minor effects were probably also involved. Induced resistance to CMV, associated with autonomously developing cell death lesions (Anl) previously not known in potato, was expressed in one parental line. The mechanisms of resistance to CMV may be associated with an inherent or developmental lack of host factors required for compatible CMV-host interactions in viral long distance transport and/or inability of CMV to efficiently suppress the host gene silencing mechanism in potatoes. Polyploidy (gene dose) and high heterozygosity (multiple homologous genes) of potato cultivars may be significant in conferring the durable resistance to CMV. These data provide explanations why CMV is not common and economically important in cultivated potatoes, even though CMV commonly occurs in other crops, weeds and wild plants in potato production areas. Received: 11 February 1999 / Accepted: 25 March 1999     

Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures

 Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397 amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity. Received: 2 September 1999 / Accepted: 30 November 1999     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

Zinc deficiency-induced phytosiderophore release by the Triticaceae is not consistently expressed in solution culture

The effects of zinc (Zn) and iron (Fe) deficiencies on phytosiderophore (PS) exudation by three barley (Hordeum vulgare L.) cultivars differing in Zn efficiency were assessed using chelator-buffered nutrient solutions. A similar study was carried out with four wheat (Triticum aestivum L. and T. durum Desf.) cultivars, including the Zn-efficient Aroona and Zn-inefficient Durati. Despite severe Zn deficiency, none of the barley or wheat cultivars studied exhibited significantly elevated PS release rates, although there was significantly enhanced PS exudation under Fe deficiency. Aroona and Durati wheats were grown in a further study of the effects of phosphate (P) supply on PS release, using 100 μM KH₂PO₄ as high P, or solid hydroxyapatite as a supply of low-release P. Phytosiderophore exudation was not increased due to P treatment under control or Zn-deficient conditions, but was increased by Fe deficiency. Accumulation of P in shoots of Zn- and Fe-deficient plants was seen in both P treatments, somewhat more so under the KH₂PO₄ treatment. Zinc-efficient wheats and grasses have been previously shown to exude more PS under Zn deficiency than Zn-inefficient genotypes. We did not observe Zn-deficiency-induced PS release and were unable to replicate the results of previous researchers. The tendency for Zn deficiency to induce PS release seems to be method dependent, and we suggest that all reported instances may be explained by an induced physiological deficiency of Fe. Received: 25 October 1999 / Accepted: 3 December 1999     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999