Distribution of indole-acetic acid, gibberellin and cytokinins in apoplast and symplast of parthenocarpic tomato fruits

The levels of indole-acetic acid (IAA), gibberellic acid1 (GA₁), trans-zeatin (Z) and trans-zeatin riboside (ZR) in seedless fruits of parthenocarpic tomato (Lycopersicon esculentum Mill. cv. Rarkuna First) were analysed using ¹³C₆-IAA, ²H₂-GA₁, ²H₅-Z and ²H₅-ZR, as internal standards by liquid chromatography–mass spectrometry. Fruits were sampled at 6 cm in diameter (referred to as 6-cm-fruit) and 8 cm (8-cm-fruit, mature green stage) and separated into pericarps, partitions and locule tissues. The pericarps and partitions were centrifuged for the collection of apoplast (AP) solution (sap outside a cell) and symplast (SP) solution (sap within a cell). IAA concentrations of the pericarps and partitions were higher in 8-cm-fruit than in 6-cm-fruit. In the partitions, IAA concentrations of SP solution were higher than those of AP solution in both 6- and 8-cm-fruit. The SP solution of the partitions in 6-cm-fruit had the highest concentration of Z (4.6 pmol/g fresh weight) and was 2.7 times than the AP solution, while in the pericarps Z concentrations were the same level in AP and SP solution. The ZR concentration in locule tissues in 6-cm-fruit (55 pmol/g fresh weight ) was the highest of all parts. The results suggest that the sites of synthesis may be the SP of partitions for IAA and Z, and locules for ZR.     

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two‐dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix‐assisted laser‐desorption ionisation time of flight mass spectrometry (MALDI ‐ TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two‐dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin‐like proteins or glucanases, and cell expansion, i.e. β‐D‐glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.     

Psychrotolerant bacteria isolated from the leaf apoplast of cold-adapted wild plants improve the cold resistance of bean (Phaseolus vulgaris L.) under low temperature

We have isolated psychrotolerant bacteria from the leaf apoplast of cold-adapted wild plants and aimed to investigate their effect on the cold resistance of bean (Phaseolus vulgaris L.). Based on the findings of 16S rRNA gene sequence analysis, 20 isolates belonging to 5 bacteria species (Pseudomonas fragi, P. chloropaphis, P. fluorescens, P. proteolytica and Brevibacterium frigoritolerans) were identified in the leaf apoplastic fluid of Draba nemorosa, Galanthus gracilis, Colchicum speciousum, Scilla siberica, Erodium cicutarium, respectively. We have determined that 6 of the 20 isolates have exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity and secreted different extracellular proteins under cold condition (+4 °C) compared to normal growth condition (28 °C). The six isolates were then inoculated independently of each other to the leaves of 10-day-old bean seedlings growing under normal conditions (25/22 °C, 16/8 h photoperiod), and the inoculated and uninoculated (control) seedlings were transferred to cold (9/5 °C, 16/8 h photoperiod) for 3 days. The bacterial inoculations have decreased freezing injury, ice nucleating activity and lipid peroxidation content in parallel with the decrease of reactive oxygen species level such as O₂^.- and H₂O₂ in the inoculated seedlings compared to the control. In addition, the inoculations of the isolates have stimulated the activity of apoplastic antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and glutathione reductase. The results show that the inoculations improve the cold resistance of bean seedlings and the psychrotolerant bacterial isolates can be evaluated within the group of plant growth promoting bacteria (PGPB) which can increase tolerance of cold-sensitive crops.     

A new application for the quantification of apoplastic redox radicals of plant roots using pre-fluorescent probe

New application of fluorescence probe to detect apoplastic redox radicals from plant roots were sought. This probe can detect radicals selectively. Calibration curve for radicals was obtained using nitrogen monoxide as radical standard produced by NOC7. Apoplastic radicals released constitutively were quantified and the release rate was 60 μmol L^?1 h^?1. Oxidative burst triggered by chitin was distinguished from constitutive radical release.     

Predawn disequilibrium between plant and soil water potentials in two cold-desert shrubs

Classical water relations theory predicts that predawn plant water potential should be in equilibrium with soil water potential (soil Ψ_w) around roots, and many interpretations of plant water status in natural populations are based on this expectation. We examined this expectation for two salt-tolerant, cold-desert shrub species in glasshouse experiments where frequent watering assured homogeneity in soil Ψ_w and soil-root hydraulic continuity and where NaCl controlled soil Ψ_w. Plant water potentials were measured with a pressure chamber (xylem Ψ_p) and thermocouple psychrometers (leaf Ψ_w). Soil Ψ_w was measured with in situ thermocouple psychrometers. Predawn leaf Ψ_w and xylem Ψ_p were significantly more negative than soil Ψ_w, for many treatments, indicating large predawn soil-plant Ψ_w disequilibria: up to 1.2 MPa for Chrysothamnus nauseosus (0 and 100 mm NaCl) and 1.8 MPa for Sarcobatus vermiculatus (0, 100, 300, and 600 mm NaCl). Significant nighttime canopy water loss was one mechanism contributing to predawn disequilibrium, assessed by comparison of xylem Ψ_p for bagged (to minimize transpiration) and unbagged canopies, and by gas exchange measurements. However, nighttime transpiration accounted for only part of the predawn disequilibrium. Other mechanisms that could act with nighttime transpiration to generate large predawn disequilibria are described and include a model of how leaf apoplastic solutes could contribute to the phenomenon. This study is among the first to conclusively document such large departures from the expectation of predawn soil-plant equilibrium for C₃ shrubs, and provides a general framework for considering relative contributions of nighttime transpiration and other plant-related mechanisms to predawn disequilibrium. Received: 12 November 1998 / Accepted: 5 May 1999     

Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: chemical nature in relation to anatomical development

The chemical nature of enzymatically isolated endodermal cell walls from Cicer arietinum L., Clivia miniata Reg. and Iris germanica L. was studied by FTIR (Fourier transform infrared) spectroscopy. Observed frequencies were assigned to functional groups present in the cell wall and relative amounts of the biopolymers suberin and lignin, cell wall carbohydrates and proteins were determined. Infrared absorption spectra indicated structural characteristics for the three different developmental states of the isolated endodermal cell wall: primary endodermis with Casparian strips (state I), secondary endodermis with suberin lamellae (state II), and tertiary endodermis with U-shaped cell wall depositions (state III). The data obtained from this study are compared with previous results obtained by chemical degradation of isolated endodermal cell walls and subsequent determination of monomeric degradation products by gas chromatography and mass spectrometry. It is concluded that FTIR spectroscopy represents a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. Furthermore, the observation that the suberin-assigned absorption bands disappeared after transesterification of the samples with BF₃-methanol confirmed that suberin is completely degraded by this treatment. Received: 20 February 1999 / Accepted: 25 May 1999     

Cascade transduction and production of the light-nitric oxide signal from shoots to roots in maize seedlings exposed to enhanced UV-B radiation

The biomasses, rate of apparent nitric oxide (NO)-release, nitric oxide synthase (NOS) activity as well as β-d-endo and exo-glucanase activity of the cell wall were analyzed and determined in the roots of maize seedlings. It was found that rhizospheric treatments of 2-phenyl-4,4,5,5-tetramethlimida-zoline-l-oxyl-3-oxide (PTIO), a NO scavenger, and radiation of enhanced ultraviolet-B (UV-B) to aerial parts of the seedling markedly inhibited the rate of NO release in roots, raised the activity of β-d-endo and exo-glucanase, and increased the biomasses of roots. The patent inhibitor, N-nitro-l-arginine (LNNA), of NOS was unable to inhibit NOS activity and NO generation. Inversely, reactive oxygen species (ROS) eliminator, N-acetyl-cysteine (NAC), stimulated the rate of NO release. There is no relationship between NOS activity and the rate of NO release. The latter showed a positive correlation with nitrate reductase (NR) activity, whereas it showed a negative correlation with the bio-masses and the activity of β-d-endo and exo-glucanase. All results implicated that NO was a by-product generated by NR catalysis, whereas NR activity was sensitively repressed by the systemic signal network (involved in ROS) induced by enhanced UV-B. It indicated that the downstream signal molecule of enhanced UV-B light is probably ROS which decreased NO generation through inhibiting NR activity. The endogenous NO generated by NR catalysis is perhaps such a messenger for restraining β-d-endo and exo-glucanase activity that the root growth was retarded.     

Active chitinases in the apoplastic fluids of healthy white lupin (Lupinus albus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupinus albus L.) plants and suspension-cultured cells by N-terminal microse-quencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lupin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.     

不同禾本科作物与花生混作对花生根系质外体铁的累积和还原力的影响

采用土培盆栽方法模拟玉米/花生、大麦/花生、燕麦/花生、小麦/花生、高粱/花生5种种植方式,研究混作对花生根系质外体铁的累积和还原力的影响.结果表明,当花生与5种分泌植物铁载体能力不同的禾本科作物混作时,花生新叶叶色正常,而单作花生则表现出严重的缺铁黄化症状,混作花生各部位的含铁量明显增加.与麦类作物(大麦、燕麦、小麦)混作的花生其各部位铁含量高于与玉米、高粱混作的花生,说明麦类作物改善花生铁营养的能力强于玉米、高粱,而两个玉米品种之间的能力差异不大,这主要是由于麦类作物分泌植物铁载体能力高于玉米、高粱.在花生生长至第50、60和70d时,混作花生根系质外体铁含量也随着逐渐增加,并始终高于单作花生.同时,混作明显地提高了花生根际土壤有效铁的含量,花生根系还原力也逐步提高.混作花生逐渐提高的还原力和介质中不断供给的易被花生还原吸收的铁,在改善花生的铁营养方面起了重要的作用.     

The measurement of water relations of plant cell culture. I. Water release in response to centrifuge-induced water potentials

Water loss by cell suspensions during centrifugation is well defined by simple physical principles. The major factors affecting water release during centrifugation are: duration of centrifogation, depth of the cell mass, density of cells, relative centripetal acceleration and centripetal force. Water release during centrifugation was best described by an exponential decay process with a decay constant that increases with acceleration from 0.31 ± 0.01 to 0.66 ± 0.12 min^?1 (mean ± SE) between 4 825 and 19 300 m s^?2, respectively. The cell mass relative water content (RWC) at equilibrium was not a function of rate of water loss and was constant for each acceleration. A centripetal force was generated by the mass of the cells being accelerated away from the axis of rotation. This force generated a pressure that removed some of the cell wall and symplast water, by compression at contact points between the cells and by compression of the cytoplasm. Pressure induced by centripetal forces ranging from ?0.02 to ?0.23 MPa gave a linear relationship (r² > 0.99) between force and RWC. The slope (0.900 MPa) was proportional to the cell wall modulus of elasticity (±). and the intercept was interpreted to give the mass of the cells at full turgor without interstitial water (RWC=1). This interpretation is supported by the findings, of two independent experiments. Centrifuged cells suspended at 100% relative humidity for over 48 h reached the same water content as predicted by the intercept. Interstitial water was labelled with solutions of polyethylene glycol (PEG. M_r 8 000), the diameter of which was too large to enter the pores of plant cell walls. Centripetal accelerations greater than 10 900 m s^?2 removed PEG-labelled water to levels below 0.9% of cell water content. Removal of interstitial water and other loosely bound water provided a convenient method for determination of growth, RWC and ±. The centrifugal methods provide the foundation for new quantitative methods for cell culture water relations analyses.