Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.).

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Phenolic acid allelochemicals induced morphological, ultrastructural, and cytological modification on Cassia sophera L. and Allium cepa L.

The allelopathic potential of leaf aqueous extract (LAE) of Calotropis procera on growth behavior, ultrastructural changes on Cassia sophera L., and cytological changes on Allium cepa L. was investigated. LAE at different concentrations (0.5, 1, 2, and 4 %) significantly reduced the root length, shoot length, and dry biomass of C. sophera. Besides, the ultrastructural changes (through scanning electron microscopy, SEM) induced in epidermal cells of 15-day-old seedlings of Cassia leaf were also noticed. The changes induced were shrinking and contraction of epidermal cells along with the formation of major grooves, canals, and cyst-like structures. The treated samples of epidermal cells no longer seem to be smooth as compared to control. LAE at different concentrations induces chromosomal aberrations and variation in shape of the interphase and prophase nucleus in A. cepa root tip cells when compared with control groups. The mitotic index in treated onion root tips decreased with increasing concentrations of the extracts. The most frequent aberrations were despiralization at prophase with the formation of micronuclei, sticky anaphase with bridges, sticky telophase, C-metaphase, etc. The results also show the induction of ghost cells, cells with membrane damage, and cells with heterochromatic nuclei by extract treatment. Upon HPLC analysis, nine phenolic acids (caffeic acid, gentisic acid, catechol, gallic acid, syringic acid, ellagic acid, resorcinol, p-coumaric acid, and p-hydroxy benzoic acid) were identified. Thus, the phenolic acids are mainly responsible for the allelopathic behavior of C. procera.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. RESULTS: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. CONCLUSION: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation.

Ammonium tolerance and toxicity of Actinoscirpus grossus--a candidate species for use in tropical constructed wetland systems.

Actinoscirpus grossus, a native species in tropical wetlands of South-East Asia, North Australia and the Pacific islands, has been reported to perform well in experimental scale constructed wetland (CW) systems. However, little is known about how high NH4(+) concentrations prevailing in wastewater affect growth and performance of this species. We examined growth, morphological and physiological responses of A. grossus to NH4(+) concentrations of 0.5, 2.5, 5, 10 and 15mM under hydroponic growth conditions. The relative growth rates (RGR) of the plants were highest at 2.5mM NH4(+) but significantly reduced at 10 and 15mM NH4(+). The roots of the plants were stunted and produced subepidermal lignified-cell layers at exposure to 10 and 15mM NH4(+). The photosynthetic rates did not differ between treatments (average An=21.3±0.4µmolCO2m(-2)s(-1)) but the photosynthetic nitrogen and carbon use efficiency (PNUE and PCUE) were significantly depressed at 10 and 15mM NH4(+) treatments. The concentration of NH4(+) in the roots, but not in the leaves, reflected the NH4(+) concentration in the growth medium suggesting that the species is unable to regulate the NH4(+) uptake. The high root respiration rates in concert with high tissue NH4(+) and declined C/N ratio at 10 and 15mM NH4(+) suggest that the NH4(+) assimilation occurs primarily in the roots and the plant has inadequate C-skeletons for NH4(+) assimilation and exudation at high NH4(+) concentration in the external solution. The concentrations of mineral cations were generally reduced and the root membrane permeability increased at high external NH4(+) concentrations. Our study shows that A. grossus tolerates NH4(+) concentrations up to 5mM which is characteristic of most types of wastewater. Hence, A. grossus is a good native candidate species for use in CW systems in tropical and subtropical climates in South-East Asia, North Australia and the Pacific islands.

Expression of abiotic stress inducible ETHE1-like protein from rice is higher in roots and is regulated by calcium.

ETHYLMALONIC ENCEPHALOPATHY PROTEIN 1 (ETHE1) encodes sulfur dioxygenase (SDO) activity regulating sulfide levels in living organisms. It is an essential gene and mutations in ETHE1 leads to ethylmalonic encephalopathy (EE) in humans and embryo lethality in Arabidopsis. At present, very little is known regarding the role of ETHE1 beyond the context of EE and almost nothing is known about factors affecting its regulation in plant systems. In this study, we have identified, cloned and characterized OsETHE1, a gene encoding ETHE1-like protein from Oryza sativa. ETHE1 proteins in general are most similar to glyoxalase II (GLYII) and hence OsETHE1 has been earlier annotated as OsGLYII1, a putative GLYII gene. Here we show that OsETHE1 lacks GLYII activity and is instead an ETHE1 homolog being localized in mitochondria like its human and Arabidopsis counterparts. We have isolated and analyzed 1618 bp OsETHE1 promoter (pOsETHE1) to examine the factors affecting OsETHE1 expression. For this, transcriptional promoter pOsETHE1: 5-bromo-5-chloro-3-indolyl-ß-D-glucuronide (GUS) fusion construct was made and stably transformed into rice. GUS expression pattern of transgenic pOsETHE1:GUS plants reveal a high root-specific expression of OsETHE1. The pOsETHE1 activity was stimulated by Ca(II) and required light for induction. Moreover, pOsETHE1 activity was induced under various abiotic stresses such as heat, salinity and oxidative stress, suggesting a potential role of OsETHE1 in stress response.

Methyl jasmonate-elicited transcriptional responses and pentacyclic triterpene biosynthesis in sweet basil.

Sweet basil (Ocimum basilicum) is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments. Although a variety of secondary metabolites with potent biological activities are identified, our understanding of the biosynthetic pathways that produce them has remained largely incomplete. We studied transcriptional changes in sweet basil after methyl jasmonate (MeJA) treatment, which is considered an elicitor of secondary metabolites, and identified 388 candidate MeJA-responsive unique transcripts. Transcript analysis suggests that in addition to controlling its own biosynthesis and stress responses, MeJA up-regulates transcripts of the various secondary metabolic pathways, including terpenoids and phenylpropanoids/flavonoids. Furthermore, combined transcript and metabolite analysis revealed MeJA-induced biosynthesis of the medicinally important ursane-type and oleanane-type pentacyclic triterpenes. Two MeJA-responsive oxidosqualene cyclases (ObAS1 and ObAS2) that encode for 761- and 765-amino acid proteins, respectively, were identified and characterized. Functional expressions of ObAS1 and ObAS2 in Saccharomyces cerevisiae led to the production of ß-amyrin and α-amyrin, the direct precursors of oleanane-type and ursane-type pentacyclic triterpenes, respectively. ObAS1 was identified as a ß-amyrin synthase, whereas ObAS2 was a mixed amyrin synthase that produced both α-amyrin and ß-amyrin but had a product preference for α-amyrin. Moreover, transcript and metabolite analysis shed light on the spatiotemporal regulation of pentacyclic triterpene biosynthesis in sweet basil. Taken together, these results will be helpful in elucidating the secondary metabolic pathways of sweet basil and developing metabolic engineering strategies for enhanced production of pentacyclic triterpenes.

Roles of BOR2, a boron exporter, in cross linking of rhamnogalacturonan II and root elongation under boron limitation in Arabidopsis.

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation.

Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton.

Nutrients such as phosphorus may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO2), which is projected to double by the end of the 21st century. Elevated CO2 may overcome the diffusional limitations to photosynthesis posed by stomata and mesophyll and alter the photo-biochemical limitations resulting from phosphorus deficiency. To evaluate these ideas, cotton (Gossypium hirsutum) was grown in controlled environment growth chambers with three levels of phosphate (Pi) supply (0.2, 0.05 and 0.01mM) and two levels of CO2 concentration (ambient 400 and elevated 800µmolmol(-1)) under optimum temperature and irrigation. Phosphate deficiency drastically inhibited photosynthetic characteristics and decreased cotton growth for both CO2 treatments. Under Pi stress, an apparent limitation to the photosynthetic potential was evident by CO2 diffusion through stomata and mesophyll, impairment of photosystem functioning and inhibition of biochemical process including the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxyganase and the rate of ribulose-1,5-bisphosphate regeneration. The diffusional limitation posed by mesophyll was up to 58% greater than the limitation due to stomatal conductance (gs) under Pi stress. As expected, elevated CO2 reduced these diffusional limitations to photosynthesis across Pi levels; however, it failed to reduce the photo-biochemical limitations to photosynthesis in phosphorus deficient plants. Acclimation/down regulation of photosynthetic capacity was evident under elevated CO2 across Pi treatments. Despite a decrease in phosphorus, nitrogen and chlorophyll concentrations in leaf tissue and reduced stomatal conductance at elevated CO2, the rate of photosynthesis per unit leaf area when measured at the growth CO2 concentration tended to be higher for all except the lowest Pi treatment. Nevertheless, plant biomass increased at elevated CO2 across Pi nutrition with taller plants, increased leaf number and larger leaf area.

Silicon supply modifies C:N:P stoichiometry and growth of Phragmites australis.

Silicon is a non-essential element for plant growth. Nevertheless, it affects plant stress resistance and in some plants, such as grasses, it may substitute carbon (C) compounds in cell walls, thereby influencing C allocation patterns and biomass production. How variation in silicon supply over a narrow range affects nitrogen (N) and phosphorus (P) uptake by plants has also been investigated in some detail. However, little is known about effects on the stoichiometric relationships between C, N and P when silicon supply varies over a broader range. Here, we assessed the effect of silicon on aboveground biomass production and C:N:P stoichiometry of common reed, Phragmites australis, in a pot experiment in which three widely differing levels of silicon were supplied. Scanning electron microscopy (SEM) showed that elevated silicon supply promoted silica deposition in the epidermis of Phragmites leaves. This resulted in altered N:P ratios, whereas C:N ratios changed only slightly. Plant growth was slightly (but not significantly) enhanced at intermediate silicon supply levels but significantly decreased at high levels. These findings point to the potential of silicon to impact plant growth and elemental stoichiometry and, by extension, to affect biogeochemical cycles in ecosystems dominated by Phragmites and other grasses and sedges.

Over-expression in the nucleotide-binding site-leucine rich repeat gene DEPG1 increases susceptibility to bacterial leaf streak disease in transgenic rice plants.

Bacterial leaf streak of rice (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a widely-spread disease in the main rice-producing areas of the world. Investigating the genes that play roles in rice-Xoc interactions helps us to understand the defense signaling pathway in rice. Here we report a differentially expressed protein gene (DEPG1), which regulates susceptibility to BLS. DEPG1 is a nucleotide-binding site (NBS)-leucine rich repeat (LRR) gene, and the deduced protein sequence of DEPG1 has approximately 64% identity with that of the disease resistance gene Pi37. Phylogenetic analysis of DEPG1 and the 18 characterized NBS-LRR genes revealed that DEPG1 is more closely related to Pi37. DEPG1 protein is located to the cytoplasm, which was confirmed by transient expression of DEPG1-GFP (green fluorescent protein) fusion construct in onion epidermal cells. Semi-quantitative PCR assays showed that DEPG1 is widely expressed in rice, and is preferentially expressed in internodes, leaf blades, leaf sheaths and flag leaves. Observation of cross sections of leaves from the transgenic plants with a DEPG1-promoter::glucuronidase (GUS) fusion gene revealed that DEPG1 is also highly expressed in mesophyll tissues where Xoc mainly colonizes. Additionally, Xoc negatively regulates expression of DEPG1 at the early stage of the pathogen infection, and so do the three defense-signal compounds including salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic-acid (ACC). Transgenic rice plants overexpressing DEPG1 exhibit enhanced susceptibility to Xoc compared to the wild-type controls. Moreover, enhanced susceptibility to Xoc may be mediated by inhibition of the expression of some SA biosynthesis-related genes and pathogenesis-related genes that may contribute to the disease resistance. Taken together, DEPG1 plays roles in the interactions between rice and BLS pathogen Xoc.

Optimization of hot water treatment for removing microbial colonies on fresh blueberry surface.

UNLABELLED: Blueberries for the frozen market are washed but this process sometimes is not effective or further contaminates the berries. This study was designed to optimize conditions for hot water treatment (temperature, time, and antimicrobial concentration) to remove biofilm and decrease microbial load on blueberries. Scanning electron microscopy (SEM) image showed a well-developed microbial biofilm on blueberries dipped in room temperature water. The biofilm consisted of yeast and bacterial cells attached to the berry surface in the form of microcolonies, which produced exopolymer substances between or upon the cells. Berry exposure to 75 and 90 °C showed little to no microorganisms on the blueberry surface; however, the sensory quality (wax/bloom) of berries at those temperatures was unacceptable. Response surface plots showed that increasing temperature was a significant factor on reduction of aerobic plate counts (APCs) and yeast/mold counts (YMCs) while adding Boxyl® did not have significant effect on APC. Overlaid contour plots showed that treatments of 65 to 70 °C for 10 to 15 s showed maximum reductions of 1.5 and 2.0 log CFU/g on APCs and YMCs, respectively; with acceptable level of bloom/wax score on fresh blueberries. This study showed that SEM, response surface, and overlaid contour plots proved successful in arriving at optima to reduce microbial counts while maintaining bloom/wax on the surface of the blueberries. PRACTICAL APPLICATION: Since chemical sanitizing treatments such as chlorine showed ineffectiveness to reduce microorganisms loaded on berry surface (Beuchat and others 2001, Sapers 2001), hot water treatment on fresh blueberries could maximize microbial reduction with acceptable quality of fresh blueberries.

Gating of aquaporins by heavy metals in Allium cepa L. epidermal cells.

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn(2+), Pb(2+), Cd(2+), Hg(2+)). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 µM-2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that ß-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20-40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating.

The Arabidopsis ATP-binding cassette protein AtMRP5/AtABCC5 is a high affinity inositol hexakisphosphate transporter involved in guard cell signaling and phytate storage.

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.

Cloning the PvP5CS gene from common bean (Phaseolus vulgaris) and its expression patterns under abiotic stresses.

A full-length cDNA denominated PvP5CS for Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in the biosynthesis of proline, was cloned from common bean using a candidate gene approach. PvP5CS contains an open reading frame encoding a 716 amino acid polypeptide. Sequence analysis showed that PvP5CS shares 95.1% homology in nucleotide sequence and 93.2% identity in amino acid sequence with the mothbean (Vigna aconitifolia) P5CS. The expression patterns of PvP5CS in common bean treated with drought, cold (4 degrees C), and salt (200 mM NaCl) stresses were examined using real-time quantitative PCR. These abiotic stresses caused significant up-regulation of the expression of PvP5CS in leaves. The PvP5CS mRNA transcript increased to 2.5 times the control level after 4d drought stress. A rapid up-regulation of PvP5CS, to about 16.3 times the control at 2h post-treatment was observed under salt stress. A significant increase in PvP5CS expression (11.7-fold) was detected after 2h of cold stress. The peaks of proline accumulation appeared at 8d for drought, 24h for cold and 9h for salt stress, somewhat later than the peaks of PvP5CS expression. These results suggest that PvP5CS was a stress-inducible gene regulating the accumulation of proline in plants subjected to stress. Finally, subcellular localization assays showed that the PvP5CS protein was present in the nucleus and at the plasmalemma.

Xyloglucan endotransglucosylase activity loosens a plant cell wall.

BACKGROUND AND AIMS: Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in the wall so that the microfibrils can move relative to each other. Here the effect was studied of a purified recombinant xyloglucan endotransglucosylase/hydrolase (XTH) on the extension of isolated cell walls. METHODS: The epidermis of growing onion (Allium cepa) bulb scales is a one-cell-thick model tissue that is structurally and mechanically highly anisotropic. In constant load experiments, the effect of purified recombinant XTH proteins of Selaginella kraussiana on the extension of isolated onion epidermis was recorded. KEY RESULTS: Fluorescent xyloglucan endotransglucosylase (XET) assays demonstrate that exogeneous XTH can act on isolated onion epidermis cell walls. Furthermore, cell wall extension was significantly increased upon addition of XTH to the isolated epidermis, but only transverse to the net orientation of cellulose microfibrils. CONCLUSIONS: The results provide evidence that XTHs can act as cell wall-loosening enzymes.

Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana.

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.

Characterization of hydrophilic and lipophilic pathways of Hedera helix L. cuticular membranes: permeation of water and uncharged organic compounds.

The permeability of astomatous leaf cuticular membranes of Hedera helix L. was measured for uncharged hydrophilic (octanol/water partition coefficient log K(O/W) < or =0) and lipophilic compounds (log K(O/W) >0). The set of compounds included lipophilic plant protection agents, hydrophilic carbohydrates, and the volatile compounds water and ethanol. Plotting the mobility of the model compounds versus the molar volume resulted in a clear differentiation between a lipophilic and a hydrophilic pathway. The size selectivity of the lipophilic pathway was described by the free volume theory. The pronounced tortuosity of the diffusional path was caused by cuticular waxes, leading to an increase in permeance for the lipophilic compounds after wax extraction. The size selectivity of the hydrophilic pathway was described by hindered diffusion in narrow pores of molecular dimensions. A distinct increase in size selectivity was observed for hydrophilic compounds with a molar volume higher than 110 cm3 mol(-1). Correspondingly, the size distribution of passable hydrophilic pathways was interpreted as a normal distribution with a mean pore radius of 0.3 nm and a standard deviation of 0.02 nm. The increased permeance of the hydrophilic compounds by the removal of cuticular waxes was attributed to an increase in the porosity, a decrease in the tortuosity, and a widening of the pore size distribution. Cuticular transpiration resulted from the permeation of water across the hydrophilic pathway. The far-reaching implications of two parallel pathways for the establishment of correlations between cuticular structure, chemistry, and function are discussed.

Somatic embryogenesis in Solanum tuberosum L.: a histological examination of key developmental stages.

A potential novel method of producing high-quality potato ( Solanum tuberosum L.) nuclear seeds is through the process of somatic embryogenesis (SE). Somatic embryo formation has been successfully reported in many plant species, but in potato, reliable SE systems are still at the experimental stage. A key factor in the success of any SE system is the ability to discriminate SE-specific cellular structures from those emerging through an organogenic route. In the investigation reported here we attempted to discriminate the progression of specific stages of potato SE by histological means. Internodal segment (INS) explants from 4- to 6-week-old cv. Desiree in vitro cultures were successively cultured on SE induction (for 2 weeks) and expression/regeneration media (for 3 weeks) with and without 2,4-dichlorophenoxyacetic acid (5 microM). Microscopic examination of histological slides prepared using INS explants at different stages revealed the presence of characteristic globular, heart and torpedo stages in the potato SE system along with other associated unique features such as protoderm development and discrete vascular connections. These results confirm the occurrence of potato SE as per the accepted definition of the term.

Evidence for symplastic involvement in the radial movement of calcium in onion roots.

The pathway of Ca(2+) movement from the soil solution into the stele of the root is not known with certainty despite a considerable body of literature on the subject. Does this ion cross an intact, mature exodermis and endodermis? If so, is its movement through these layers primarily apoplastic or symplastic? These questions were addressed using onion (Allium cepa) adventitious roots lacking laterals. Radioactive Ca(2+) applied to the root tip was not transported to the remainder of the plant, indicating that this ion cannot be supplied to the shoot through this region where the exodermis and endodermis are immature. A more mature zone, in which the endodermal Casparian band was present, delivered 2.67 nmol of Ca(2+) mm(-1) treated root length d(-1) to the transpiration stream, demonstrating that the ion had moved through an intact endodermis. Farther from the root tip, a third zone in which Casparian bands were present in the exodermis as well as the endodermis delivered 0.87 nmol Ca(2+) mm(-1) root length d(-1) to the transpiration stream, proving that the ion had moved through an unbroken exodermis. Compartmental elution analyses indicated that Ca(2+) had not diffused through the Casparian bands of the exodermis, and inhibitor studies using La(3+) and vanadate (VO(4)(3-)) pointed to a major involvement of the symplast in the radial transport of Ca(2+) through the endodermis. It was concluded that in onion roots, the radial movement of Ca(2+) through the exodermis and endodermis is primarily symplastic.