Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated "symplastic" phloem loaders, as opposed to "apoplastic" loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

Use of CE to Analyze Solutes in Pico- and Nano-Liter Samples from Plant Cells and Rhizosphere.

This chapter describes the use of capillary electrophoresis (CE) in the accurate quantitative mapping of small molecules and ions in intact function tissues between individual cells at single cell resolution. It can also be used for the analysis of the heterogeneity of soil surrounding roots at similar spatial resolution, providing a link between plant and environment. No pretreatment or genetic manipulation of the plant is required. The application is an extension of the Single Cell Sampling and Analysis technique (SiCSA), in which glass micromanipulation of microcapillaries allows samples in the pl and nl volume range to be obtained and manipulated under paraffin oil (to prevent evaporation) before being introduced to the CE column. An advantage of this approach is that the entire sample can be brought to the detector (without the loading losses associated with other techniques). The power of SiCSA-CE is that the results can be directly related to a range of other single-cell resolution parameters ranging from mechanical and hydraulic properties to gene expression. Several protocols and (contrasting) applications are provided.

Aluminium-induced alteration of ion homeostasis in root tip vacuoles of two maize varieties differing in Al tolerance.

Root elongation is a primary target of Al toxicity in plants. The objective of this study was to see whether Al-induced disturbance of ion homeostasis is related to the inhibition of root elongation. For this purpose, root growth rate, free cytoplasmic calcium (Ca²+) and vacuolar content of phosphate (P(i)), potassium (K+), nitrate (NO3⁻) and malate, as well as malate and citrate exudation and nitrate reductase activity were analysed in tips of two Zea mays L. varieties differing in Al resistance. Aluminium treatment affected root growth and cytoplasmic Ca²+ in the Al sensitive variety Bakero, but not in the Al tolerant variety Sikuani. However, both varieties suffered Al-induced decrease of vacuolar K+, and phosphate concentrations. Vacuolar malate concentrations were more affected by Al in Bakero than in Sikuani. Vacuolar nitrate concentrations increased upon Al exposure in both varieties. Only in Sikuani rhizosphere, pH slightly increased upon Al exposure. Our data are consistent with the hypothesis that disturbance of Ca²+ homeostasis is an early event in the Al toxicity syndrome. However, Al-induced alterations of the root tip homeostasis of major ions seem unrelated to Al-induced inhibition of root elongation.

Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis.

BACKGROUND: Holistic profiling and systems biology studies of nutrient availability are providing more and more insight into the mechanisms by which gene expression responds to diverse nutrients and metabolites. Less is known about the mechanisms by which gene expression is affected by endogenous metabolites, which can change dramatically during development. Multivariate statistics and correlation network analysis approaches were applied to non-targeted profiling data to investigate transcriptional and metabolic states and to identify metabolites potentially influencing gene expression during the heterotrophic to autotrophic transition of seedling establishment. RESULTS: Microarray-based transcript profiles were obtained from extracts of Arabidopsis seeds or seedlings harvested from imbibition to eight days-old. 1H-NMR metabolite profiles were obtained for corresponding samples. Analysis of transcript data revealed high differential gene expression through seedling emergence followed by a period of less change. Differential gene expression increased gradually to day 8, and showed two days, 5 and 7, with a very high proportion of up-regulated genes, including transcription factor/signaling genes. Network cartography using spring embedding revealed two primary clusters of highly correlated metabolites, which appear to reflect temporally distinct metabolic states. Principle Component Analyses of both sets of profiling data produced a chronological spread of time points, which would be expected of a developmental series. The network cartography of the transcript data produced two distinct clusters comprising days 0 to 2 and days 3 to 8, whereas the corresponding analysis of metabolite data revealed a shift of day 2 into the day 3 to 8 group. A metabolite and transcript pair-wise correlation analysis encompassing all time points gave a set of 237 highly significant correlations. Of 129 genes correlated to sucrose, 44 of them were known to be sucrose responsive including a number of transcription factors. CONCLUSIONS: Microarray analysis during germination and establishment revealed major transitions in transcriptional activity at time points potentially associated with developmental transitions. Network cartography using spring-embedding indicate that a shift in the state of nutritionally important metabolites precedes a major shift in the transcriptional state going from germination to seedling emergence. Pair-wise linear correlations of transcript and metabolite levels identified many genes known to be influenced by metabolites, and provided other targets to investigate metabolite regulation of gene expression during seedling establishment.

Long-distance movement of Cauliflower mosaic virus and host defence responses in Arabidopsis follow a predictable pattern that is determined by the leaf orthostichy.

Long-distance virus transport takes place through the vascular system and is dependent on the movement of photoassimilates. Here, patterns of symptom development, virus movement and gene expression were analysed in Arabidopsis following inoculation with Cauliflower mosaic virus (CaMV) on a single leaf. Virus accumulation and expression of markers for the salicylic acid (SA) and ethylene/jasmonate (Et/JA) defence pathways, PR-1 and PDF1.2, were analysed on a leaf-by-leaf basis by real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus spread followed a strictly defined pattern identical to that of a source-sink relationship. This was exploited to study differences between local and systemic defence responses in a developmental and spatial manner. In infected plants, PR-1 transcripts accumulated primarily but not exclusively in leaves with a direct vascular connection to the inoculated leaf. Abundances fell significantly as virus accumulated. By contrast, PDF1.2 transcripts were significantly lower than in controls in all leaves at early stages of infection, but recovered as virus accumulated. Virus and PR-1 transcript abundances are negatively correlated, and SA- and Et/JA-mediated signalling of gene expression occurs independently of the presence of virus. Although SA-dependent signalling responses were mainly linked to the orthostichy, Et/JA-dependent responses were independent of vascular connections.

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement.

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.

Turgor, solute import and growth in maize roots treated with galactose.

It has been observed that extension growth in maize roots is almost stopped by exposure to 5 mm d-galactose in the root medium, while the import of recent photoassimilate into the entire root system is temporarily promoted by the same treatment. The aim of this study was to reconcile these two apparently incompatible observations. We examined events near the root tip before and after galactose treatment since the tip region is the site of elongation and of high carbon deposition in the root. The treatment rapidly decreased root extension along the whole growing zone. In contrast, turgor pressure, measured directly with the pressure probe in the cortical cells of the growing zone, rapidly increased by 0.15 MPa within the first hour following treatment, and the increase was maintained over the following 24 h. Both tensiometric measurements and a comparison of turgor pressure with local growth rate demonstrated that a rapid tightening of the cell wall caused the reduction in growth. Single cell sampling showed cell osmotic pressure increased by 0.3 MPa owing to accumulation of both organic and inorganic solutes. The corresponding change in cell water potential was a rise from -0.18 MPa to approximately zero. More mature cells at 14 mm from the root tip (just outside the growing region) showed a qualitatively similar response.

Biosensor reporting of root exudation from Hordeum vulgare in relation to shoot nitrate concentration.

The aim of this study was to determine the relationship between shoot nitrate concentration, mediated by nitrate supply to roots, and root exudation from Hordeum vulgare. Plants were grown for 14 d in C-free sand microcosms, supplied with nutrient solution containing 2 mM nitrate. After this period, three treatments were applied for a further 14 d: (A) continued supply with 2 mM nitrate (zero boost), (B) supply with 10 mM nitrate (low boost), and (C) supply with 20 mM nitrate (high boost). At the end of the treatment period, a bacterial biosensor (Pseudomonas fluorescens 10586 pUCD607, marked with the lux CDABE genes for bioluminescence) was applied to the microcosms to report on C-substrate availability, as a consequence of root exudation. The nitrate boost treatments significantly affected shoot nitrate concentrations, in the order C>B>A. In treatments receiving a nitrate boost (B, C), increased shoot nitrate concentration was correlated with increased plant biomass, reduced root length, reduced number of root tips, and increased mean root diameter, relative to the no boost treatment (A). Imaging of biosensor bioluminescence (proportional to metabolic activity in response to availability of root exudates) indicated that root exudation increased with decreasing shoot nitrate concentration. Biosensor reporting of root C-flow indicated that exudation was greater from root tip regions than from the whole root, but that specific exudation rates for all sites were unaffected by treatments. Total root exudation across treatments was found to be closely correlated with total root length, indicating that increased root exudation, per unit root biomass, with decreasing nitrate supply was associated with altered root morphology, as a consequence of systemic plant responses to internal N-status.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

Changes in osmotic and turgor pressure in response to sugar accumulation in barley source leaves.

Pressure-probe measurements and single-cell sampling and analysis techniques were used to determine the effect of photosynthetic production and accumulation of sugars on osmotic and turgor pressures of individual cells of barley ( Hordeum vulgare L.) source leaves. In control plants, the changes in osmotic pressure in individual cells during the photoperiod were different for mesophyll (increase of 276 mOsmol/kg), parenchymatous bundle sheath (PBS; increase of 100 mOsmol/kg) and epidermis (remains constant). There was also an increase in osmotic pressure at the tissue level. Cooling of roots and the shoot apical meristem restricted the export of sugars from leaves, and the resulting changes in osmotic and turgor pressure were monitored. In contrast to the control leaves, mesophyll, PBS, and epidermal cells showed a similar increase in osmotic pressure (up to 500 mOsmol/kg). Cooling also increased the turgor pressure in epidermal and (to a greater extent) PBS cells. The difference in turgor pressure between epidermal and PBS cells is consistent with the presence of a water potential gradient within the leaf, from the vascular bundles towards the leaf surface.

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells.

The contents of single plant cells can be sampled using glass microcapillaries. By combining such single-cell sampling with reverse transcription-polymerase chain reaction (RT-PCR), transcripts of individual genes can be identified and, in principle, quantified. This provides a valuable technique for the analysis and quantification of the intercellular distribution of gene expression in complex tissues. In a proof-of-principle study, the cellular locations of the transcripts of the eight isoforms of actin ( ACT) expressed in Arabidopsis thaliana (L.) Heynh. were analyzed. Cell sap was extracted from epidermal and mesophyll cells of leaves of 3- to 4-week-old plants. Single-cell (SC)-RT-PCR was used to amplify the actin transcripts using specific primer pairs for ACT1, 2, 3, 4, 7, 8, 11 and 12. Only ACT2 and ACT8 were found in epidermal and in mesophyll cells. In individual trichomes, in addition to ACT2 and ACT8, ACT7 and ACT11 transcripts were detectable. By employing the already well-characterized actin system we demonstrate the practicality and power of SC-RT-PCR as a technique for analyzing gene expression at the ultimate level of resolution, the single cell.

THE PRESSURE PROBE: A Versatile Tool in Plant Cell Physiology.

This review discusses how the pressure probe has evolved from an instrument for measuring cell turgor and other water relations parameters into a device for sampling the contents of individual higher plant cells in situ in the living plant. Together with a suite of microanalytical techniques it has permitted the mapping of water and solute relations at the resolution of single cells and has the potential to link quantitatively the traditionally separate areas of water relations and metabolism. The development of the probe is outlined and its modification to measure root pressure and xylem tension described. The deployment of the pressure probe to determine and map turgor, hydraulic conductivity, reflection coefficient, cell rheological properties, solute concentrations and enzyme activities at the resolution of single cells is discussed. The controversy surrounding the interpretation of results obtained with the xylem-pressure probe is included. Possible further developments of the probe and applications of single cell sampling are suggested.