Showing papers in "Planta in 2001"

Molecular mechanisms of plant metal tolerance and homeostasis.

Abstract: Transition metals such as copper are essential for many physiological processes yet can be toxic at elevated levels. Other metals (e.g. lead) are nonessential and potentially highly toxic. Plants – like all other organisms – possess homeostatic mechanisms to maintain the correct concentrations of essential metal ions in different cellular compartments and to minimize the damage from exposure to nonessential metal ions. A regulated network of metal transport, chelation, trafficking and sequestration activities functions to provide the uptake, distribution and detoxification of metal ions. Some of the components of this network have now been identified: a number of uptake transporters have been cloned as well as candidate transporters for the vacuolar sequestration of metals. Chelators and chaperones are known, and evidence for intracellular metal trafficking is emerging. This recent progress in the molecular understanding of plant metal homeostasis and tolerance is reviewed.

Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses

Abstract: The capacity for photosynthetic acclimation in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was assessed during growth over a broad range of irradiance. Discontinuities in the response to growth irradiance were revealed for the light- and CO2-saturated rate of photosynthesis (Pmax) and the ratio of chlorophyll a to chlorophyll b (Chl a/b). Three separate phases in the response of Pmax and Chl a/b to growth light were evident, with increases at low and high irradiance ranges and a plateau at intermediate irradiance. By measuring all chlorophyll-containing components of the thylakoid membrane that contribute to Chl a/b we reveal that distinct strategies for growth at low and high irradiance underlie the discontinuous response. These strategies include, in addition to changes in the major light-harvesting complexes of photosystem II (LHCII), large shifts in the amounts of both reaction centres as well as significant changes in the levels of minor LHCII and LHCI components.

Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers

Abstract: Plants are exposed to a wide range of toxic and bioactive low-molecular-weight molecules from both exogenous and endogenous sources. Glycosylation is one of the primary sedative mechanisms that plants utilise in order to maintain metabolic homeostasis. Recently, a range of glycosyltransferases has been characterized in detail with regard to substrate specificity. The next step in increasing our understanding of the biology of glycosylation will require information regarding the exact role of individual glycosyltransferases in planta, as well as an insight into their potential involvement in metabolon-complexes. Hopefully, this will answer how a large number of glycosyltransferases with broad, rather than narrow, substrate specificity can be constrained in order to avoid interfering with other pathways of primary and secondary metabolism. These and other topics are discussed.

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth

Abstract: Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg-1 dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO2 atmosphere (4,000 microliters CO2 l-1), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO2 at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape.

The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants.

Abstract: The npq1 mutant of Arabidopsis thaliana (L.) Heynh. has no xanthophyll cycle due to a lack of functional violaxanthin de-epoxidase. Short-term exposure (<2 days) of detached leaves or whole plants to the combination of high photon flux density (1,000 µmol m–2 s–1) and low temperature (10 °C) resulted in PSII photoinhibition which was more acute in npq1 than in the wild type. This increased photosensitivity of npq1 at chilling temperature was attributable to the inhibition of nonphotochemical energy quenching (NPQ) and not to the absence of zeaxanthin itself. In contrast to PSII, PSI was found to be phototolerant to chilling stress in the light in both genotypes. In the long term (10–12 days), PSII activity recovered in both npq1 and wild type, indicating that A. thaliana is able to acclimate to chilling stress in the light independently of the xanthophyll cycle. In npq1, photoacclimation involved a substantial reduction of the light-harvesting pigment antenna of PSII and an improvement of photosynthetic electron transport. Chilling stress also induced synthesis of early light-induced proteins (ELIPs) which, in the long term, disappeared in npq1 and remained stable in the wild type. In both genotypes, photoacclimation at low temperature induced the accumulation of various antioxidants including carotenoids (except β-carotene), vitamin E (α- and γ-tocopherol) and non-photosynthetic pigments (anthocyanins and other flavonoids). Analysis of flavonoid-deficient tt mutants revealed that UV/blue-light-absorbing flavonols have a strong protective function against excess visible radiations. In contrast to the defect in npq1, the absence of flavonoids could not be overcome in the long term by compensatory mechanisms, leading to extensive photooxidative and photoinhibitory damage to the chloroplasts. Depth profiling of the leaf pigments by phase-resolved photoacoustic spectroscopy showed that the flavonoid-related photoprotection was due to light trapping, which decreased chlorophyll excitation by blue light. In contrast to flavonoids, the xanthophyll cycle and the associated NPQ seem to be mainly relevant to the protection of photosynthesis against sudden increases in light intensity.

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite.

Abstract: Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation.

Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.)

Abstract: Aerenchyma is a tissue type characterised by prominent intercellular spaces which enhance flooding tolerance in some plant species by facilitating gas diffusion between roots and the aerial environment. Aerenchyma in maize roots forms by collapse and death of some of the cortical cells in a process that can be promoted by imposing oxygen shortage or by ethylene treatment. Maize roots grown hydroponically in 3% oxygen, 1 microl x l(-1) ethylene or 21% oxygen (control) were analysed by a combination of light and electron microscopy. Use of in-situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) suggested internucleosomal cleavage of DNA. However, chromatin condensation detectable by electron microscopy was preceded by cytoplasmic changes including plasma membrane invagination and the formation of vesicles, in contrast to mammalian apoptosis in which chromatin condensation is the first detectable event. Later, cellular condensation, condensation of chromatin and the presence of intact organelles surrounded by membrane resembling apoptotic bodies were observed. All these events were complete before cell wall degradation was apparent. Therefore, aerenchyma formation initiated by hypoxia or ethylene appears to be a form of programmed cell death that shows characteristics in part resembling both apoptosis and cytoplasmic cell death in animal cells.

Identification of a transcription factor specifically expressed at the onset of leaf senescence.

Abstract: The differential expression of genes was analyzed during leaf senescence in Arabidopsis thaliana (L.) Heynh., using suppression subtractive hybridization (SSH). In order to characterize the differential expression of regulatory genes, the analysis was performed at a very early time point when leaves first differed in their photochemical efficiency (Fv/Fm) and cab transcript levels, but no visible sign of senescence, and no expression of SAG12 could be determined. After high-throughput screening, we isolated several differentially expressed cDNA clones, including a transcription factor of the WRKY family, WRKY53. All family members contained the WRKY domain, a 60-amino-acid domain with the conserved WRKYGQK motif at the N-terminal end, together with a novel zinc-finger motif. The mRNA level of WRKY53 increased substantially within the rosette leaves of a 6-week-old plant before the expression of SAG12 became detectable, was constant in all leaves of a 7-week-old plant and decreased again in 8-week-old plants. This indicates that WRKY53 is expressed at a very early time point of leaf senescence and might therefore play a regulatory role in the early events of leaf senescence.

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes

Abstract: In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes.

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

Abstract: The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight.

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells.

Abstract: The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae).

Abstract: Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives.Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands.

The physiology of ozone induced cell death.

Abstract: Ozone (O3) is a toxic air pollutant that is, somewhat paradoxically, both beneficial and harmful to life on earth. While stratospheric O3 shields biologically harm ful UY radiation from reaching the earth's surface, tropospheric 03 is toxic to biological organisms. The discovery of the phytotoxicity of O3 during the mid 1950s (Richards et al. 1958) prompted widespread studies on the effects of O3 on plant growth and development. The effect of 03 on biological organisms is attributed to its ability to spontaneously dismutate or react with cellular constituents to generate excess active oxygen species (AOS; Rao et al. 2000a). Several excellent reviews that discuss the source and the chemical reactions of 03 for mation, and the physiological effects of 03 on flora are available, and will not be discussed here (Darrall 1989; Heath and Taylor 1997; Pell et al. 1997). Instead, recent advances on the complex signal transduction pathways of 03-induced cell death will be reviewed.

NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay.

Abstract: Using the tetrazolium salt XTT (Na,3′-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O2 ·−) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O2 ·− in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O2 ·−-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O2 ·−-producing activity of coleoptiles. The maize NADH-dependent O2 ·−-synthase activity could clearly be differentiated from peroxidase-mediated O2 ·−-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of O2 ·−, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O2 ·− and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.

Non-photosynthetic enhancement of growth by high CO2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta).

Abstract: The effects of increased CO2 levels (10,000 μl l−1) in cultures of the green nitrophilic macroalga Ulva rigida C. Agardh were tested under conditions of N saturation and N limitation, using nitrate as the only N source. Enrichment with CO2 enhanced growth, while net photosynthesis, gross photosynthesis, dark respiration rates and soluble protein content decreased. The internal C pool remained constant at high CO2, while the assimilated C that was released to the external medium was less than half the values obtained under ambient CO2 levels. This higher retention of C provided the source for extra biomass production under N saturation. In N-sufficient thalli, nitrate-uptake rate and the activity of nitrate reductase (EC 1.6.6.1) increased under high CO2 levels. This did not affect the N content or the internal C:N balance, implying that the extra N-assimilation capacity led to the production of new biomass in proportion to C. Growth enhancement by increased level of CO2 was entirely dependent on the enhancement effect of CO2 on N-assimilation rates. The increase in nitrate reductase activity at high CO2 was not related to soluble carbohydrates or internal C. This indicates that the regulation of N assimilation by CO2 in U. rigida might involve a different pathway from that proposed for higher plants. The role of organic C release as an effective regulatory mechanism maintaining the internal C:N balance in response to different CO2 levels is discussed.

The influence of intact-plant and excised-leaf bioassay designs on volicitin- and jasmonic acid-induced sesquiterpene volatile release in Zea mays.

Abstract: Induced plant responses to insect attack include the release of volatile chemicals. These volatiles are used as host-location signals by foraging parasitoids, which are natural enemies of insect herbivores. A plant's response to herbivory can be influenced by factors present in insect oral secretions. Volicitin (N-(17-hydroxylinolenoyl)-L-glutamine), identified in beet armyworm (Spodoptera exigua) oral secretions, stimulates volatile release in corn (Zea mays L.) seedlings in a manner similar to beet armyworm herbivory. Volicitin is hypothesized to trigger release of induced volatiles, at least in part, by modulating levels of the wound hormone, jasmonic acid (JA). We compare the sesquiterpene volatile release of damaged leaves treated with aqueous buffer only or with the same buffer containing volicitin or JA. Leaves were damaged by scratching with a razor and test solutions were applied to the scratched area. The leaves were either excised from the plant or left intact shortly after this treatment. Plants were treated at three different times (designated as Evening, Midnight, and Morning) and volatiles were collected in the subsequent photoperiod. JA and volicitin treatments stimulated the release of volatile sesquiterpenes, namely β-caryophyllene, (E)-α-bergamotene, and (E)-β-farnesene. In all cases, JA stimulated significant sesquiterpene release above mechanical damage alone. Volicitin induced an increase in sesquiterpene volatiles for all excised-leaf bioassays and the Midnight intact plants. Volicitin treatments in the Evening and Morning intact plants produced more sesquiterpenes than the untreated controls, while mechanical damage alone produced an intermediate response that did not differ from either treatment group. Excised leaves produced a 2.5- to 8.0-fold greater volatile response than similarly treated intact plants. Excision also altered the ratio of JA-and volicitin-induced sesquiterpene release by preferentially increasing (E)-β-farnesene levels relative to β-caryophyllene. The inducibility of volatile release varied with time of treatment. On average, sesquiterpene release was highest in the Midnight excised leaves and lowest in the Morning intact plants. The duration of induced volatile release also differed between treatments. On average, JA produced a sustained release of sesquiterpenes over time, with over 20% of the combined sesquiterpenes released in the third and final volatile collection period. In contrast, less than 8% of the combined sesquiterpenes induced by volicitin were emitted during this period. The large quantitative differences between intact plants and detached leaves suggest that the results of assays using excised tissues should be cautiously interpreted when considering intact-plant models.

First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli

Abstract: For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba × Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases. Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein.

Movement and regeneration of epicuticular waxes through plant cuticles

Abstract: Regeneration of plant epicuticular waxes was studied in 24 plant species by high-resolution scanning electron microscopy. According to their regeneration behaviour, four groups could be distinguished: (i) regeneration occurs at all stages of development; (ii) regeneration occurs only during leaf expansion; (iii) regeneration occurs only in fully developed leaves; (iv) plants were not able to regenerate wax. Wax was removed from the leaves with water-based glue and a liquid polymer, i.e. water-based polyurethane dispersion. In young leaves these coverings could not be removed without damaging the leaves. After a few days, waxes appeared on the surface of these polymer films, which still adhered to the leaves. It is concluded that waxes move through the cuticle in a process similar to steam distillation. This hypothesis could be further substantiated in refined in vitro experiments. Wax isolated from Eucalyptus globulus was applied to a filter paper, subsequently covered with a liquid polymer and fixed onto a diffusion chamber filled with water. The diffusion chamber was put into a desiccator. After 8-10 days at room temperature, crystals similar in dimensions and shape to in situ crystals appeared on the surface of the polyurethane film. This indicates that waxes in molecular dimensions move together with the water vapor that permeates through the polymer membrane. Based on these results, we propose a new and simple hypothesis for the mechanism of wax movement: the molecules that finally form the epicuticular wax crystals are moved in the cuticular water current.

Expression patterns of cell wall-modifying enzymes during grape berry development

Abstract: During ripening of grape (Vitis vinifera L.) berries, softening occurs concomitantly with the second growth phase of the fruit and involves significant changes in the properties of cell wall polysaccharides. Here, the activities of enzymes that might participate in cell wall modification have been monitored throughout berry development. α-Galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23) and pectin methylesterase (EC 3.1.1.11) activities were present, but no polygalacturonase (EC 3.2.1.15), cellulase (EC 3.2.1.4), xyloglucanase (xyloglucan-specific cellulase EC 3.2.1.4) or galactanase (EC 3.2.1.89) could be detected. The accumulation of mRNAs encoding wall-modifying enzymes was examined by northern hybridization analysis. Transcripts for β-galactosidase, pectin methylesterase, polygalacturonase, pectate lyase (EC 4.2.2.2) and xyloglucan endotransglycosylase (EC 2.4.1.207) were present during ripening, although polygalacturonase activity had not been detected in berry extracts. Cellulases could not be detected in ripening berries, either at the enzyme or mRNA levels. The increase in β-galactosidase activity and mRNA is consistent with the observed decrease in type-I arabinogalactan content of the walls during ripening, and the detection of polygalacturonase and pectate lyase mRNAs might explain the increased solubility of galacturonan in walls of ripening grapes. Thus, the modification of cell wall polysaccharides during softening of grape berries is a complex process involving subtle changes to different components of the wall, and in many cases only small amounts of enzyme activity are required to effect these changes.

Early primordium morphogenesis during lateral root initiation in Arabidopsis thaliana.

Abstract: The first morphogenetic events of lateral root primordium (LRP) formation in the Arabidopsis thaliana (L.) Heynh. pericycle occur soon after cells of the primary root complete elongation. Pericycle cells in direct contact with underlying protoxylem cells participate in LRP formation. Two types of LRP initiation were found, longitudinal uni- and bi-cellular. These occur when a single or two pericycle cells within a file, respectively, become founder cells for the entire longitudinal extent of the LRP. Histochemical and cytological analysis suggests that three is the minimum number of cells required to initiate an LRP. In young primordia comprising less than 32 cells, the average cell-doubling time was 3.7 h, indicating a drastic acceleration of cell cycle progression after lateral root initiation. Early in LRP development, cell growth is limited and therefore cytokinesis leads to a reduction of cell volume, similar to cleavage division cycles during animal and plant embryogenesis. The striking coordination of proliferation between pericycle cells in adjacent files in direct contact with the underlying protoxylem implies that intercellular signaling mechanisms act in the root apical meristem or later in development.

In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana.

Abstract: Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis.

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family

Abstract: Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK ( omatic mbryogenesis eceptor-like inase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster.

Abstract: Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.

Photoinactivation of photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves.

Abstract: Leaf segments from Capsicum annuum plants grown at 100 μmol photons m−2 s−1 (low light) or 500 μmol photons m−2 s−1 (high light) were illuminated at three irradiances and three temperatures for several hours. At various times, the remaining fraction (f) of functional photosystem II (PS II) complexes was measured by a chlorophyll fluorescence parameter (1/Fo− 1/Fm, where Fo and Fm are the fluorescence yields corresponding to open and closed PS II traps, respectively), which was in turn calibrated by the oxygen yield per saturating single-turnover flash. During illumination of leaf segments in the presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the decline of f from 1.0 to about 0.3 was mono-exponential. Thereafter, f declined much more slowly, the remaining fraction (≈0.2) being able to survive prolonged illumination. The results can be interpreted as being in support of the hypothesis that photoinactivated PS II complexes photoprotect functional neighbours (G. Oquist et al. 1992, Planta 186: 450–460), provided it is assumed that a photoinactivated PS II is initially only a weak quencher of excitation energy, but becomes a much stronger quencher during prolonged illumination when a substantial fraction of PS II complexes has also been photoinactivated. In the absence of lincomycin, photoinactivation and repair of PS II occur in parallel, allowing f to reach a steady-state value that is determined by the treatment irradiance, temperature and growth irradiance. The results obtained in the presence and absence of lincomycin are analysed according to a simple kinetic model which formally incorporates a conversion from weak to strong quenchers, yielding the rate coefficients of photoinactivation and of repair for various conditions, as well as gaining an insight into the influence of f on the rate coefficient of photoinactivation. They demonstrate that the method is a convenient alternative to the use of radiolabelled amino acids for quantifying photoinactivation and repair of PS II in leaves.

Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

Abstract: The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 µM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.

The nitrogen content of the tomato leaf apoplast increases during infection by Cladosporium fulvum

Abstract: To address the problem of the nutritional requirements of phyto-pathogenic fungi growing in planta, the environment for the intercellular biotrophic pathogen, Cladosporium fulvum Cooke, of tomato (Lycopersicon esculentum Mill.) was analysed. Using a novel technique for infiltrating the intercellular space, we measured the concentrations of 21 amino acids, nitrate and ammonia in the apoplast of the tomato leaf during infection. The concentrations of most amino acids, and total nitrogen content, increased during infection. The levels of nearly all amino acids remained relatively unchanged during an incompatible interaction. All protein amino acids were detected during infection, except cysteine and tryptophan. Most amino acids were present at a concentration between 0.1–0.7 mM. The non-protein amino acid γ-aminobutyric acid was detected at the highest concentration (up to 2.5 mM) during the compatible interaction. Preliminary investigations on the source of the amino acids revealed that protease activity within the apoplast increased during infection and that infection induced the expression of the pathogenicity-related extracellular serine protease P69B. The nitrogen status of the infecting fungus and sources for the additional amino acids are discussed.

Regulatory network of tetrapyrrole biosynthesis – studies of intracellular signalling involved in metabolic and developmental control of plastids

Abstract: Plant tetrapyrrole metabolism is located in two different organelles and distributes end products into the whole cell. A complex regulatory network is involved to prevent metabolic imbalance and inefficient allocation of intermediates as well as to correlate the metabolic activities with organelle development. This review presents new findings about the control of tetrapyrrole biosynthesis and addresses the question of which regulatory principles are involved in controlling the expression of the participating enzymes and the metabolic flow in the entire pathway. It is suggested that functional organelles are required for nuclear gene expression and that metabolic signals participate in a signalling cascade transferring information from plastids to the nucleus. Recent reports about plastid-localised control mechanisms for plant tetrapyrrole metabolism are summarised and compared with results obtained in experiments on nucleus-plastid communication.

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model.

Abstract: Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784-8789). In the present study, two possible intermediates in the synthesis of violaxanthin from beta-carotene were identified in P. tricornutum, namely beta-cryptoxanthin and beta-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light beta-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kuitzing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, beta-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms.

Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways.

Abstract: Seed germination of Nicotiana tabacum L. cv. Havana 425 is determined by the balance of forces between the growth potential of the embryo and the mechanical restraint of the micropylar endosperm. In contrast to the gibberellin GA4, the brassinosteroid (BR) brassinolide (BL) did not release photodormancy of dark-imbibed photodormant seeds. Brassinolide promoted seedling elongation and germination of non-photodormant seeds, but did not appreciably affect the induction of class I β-1,3-glucanase (βGLU I) in the micropylar endosperm. Brassinolide, but not GA4, accelerated endosperm rupture of tobacco seeds imbibed in the light. Brassinolide and GA4 promoted endosperm rupture of dark-imbibed non-photodormant seeds, but only GA4 enhanced βGLU I induction. Promotion of endosperm rupture by BL was dose-dependent and 0.01 µM BL was most effective. Brassinolide and GA4 promoted abscisic acid (ABA)-inhibited dark-germination of non-photodormant seeds, but only GA4 replaced light in inducing βGLU I. These results indicate that BRs and GAs promote tobacco seed germination by distinct signal transduction pathways and distinct mechanisms. Gibberellins and light seem to act in a common pathway to release photodormancy, whereas BRs do not release photodormancy. Induction of βGLU I in the micropylar endosperm and promotion of release of 'coat-enhanced' dormancy seem to be associated with the GA-dependent pathway, but not with BR signalling. It is proposed that BRs promote seed germination by directly enhancing the growth potential of the emerging embryo in a GA- and βGLU I-independent manner.

Phosphate status affects the gene expression, protein content and enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis.

Abstract: Phosphate status affects the gene expression, protein content and enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis.