Combining Heat Stress with Pre-Existing Drought Exacerbated the Effects on Chlorophyll Fluorescence Rise Kinetics in Four Contrasting Plant Species.

Although drought and high temperature are two main factors affecting crop productivity and forest vegetation dynamics in many areas worldwide, little work has been done to describe the effects of heat combined with pre-existing drought on photochemical function in diverse plant species. This study investigated the biophysical status of photosystem II (PSII) and its dynamic responses under 2-day heat stress during a 2-week drought by measuring the polyphasic chlorophyll fluorescence rise (OJIP) kinetics. This study examined four contrasting species: a C3 crop/grass (wheat), a C4 crop/grass (sorghum), a temperate tree species (Fraxinus chinensis) and a tropical tree species (Radermachera sinica). Principal component analysis showed that the combination of heat and drought deviated from the effect of heat or drought alone. For all four species, a linear mixed-effects model analysis of variance of the OJIP parameters showed that the deviation arose from decreased quantum yield and increased heat dissipation of PSII. The results confirmed, in four contrasting plant species, that heat stress, when combined with pre-existing drought, exacerbated the effects on PSII photochemistry. These findings provide direction to future research and applications of chlorophyll fluorescence rise OJIP kinetics in agriculture and forestry, for facing increasingly more severe intensity and duration of both heat and drought events under climate change.

Involvement of SUT1 and SUT2 Sugar Transporters in the Impairment of Sugar Transport and Changes in Phloem Exudate Contents in Phytoplasma-Infected Plants.

Phytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. However, how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomatoes infected by "Candidatus Phytoplasma solani" at the beginning of the symptomatic stage, the first symptoms appearing in the newly emerged leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. In symptomatic sink leaves, leaf curling was associated with higher starch accumulation and the expression of defense genes. The analysis of leaf midribs of symptomatic leaves indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism differed from these in noninfected plants. The phytoplasma also multiplied in the three lower source leaves, even if it was not associated with the symptoms. In these leaves, the rate of phloem sucrose exudation was lower for infected plants. Metabolite profiling of phloem sap-enriched exudates revealed that glycolate and aspartate levels were affected by the infection. Their levels were also affected in the noninfected SUT1- and SUT2-antisense lines. The findings suggest the role of sugar transporters in the responses to infection and describe the consequences of impaired sugar transport on the primary metabolism.

Salinity Effects on Sugar Homeostasis and Vascular Anatomy in the Stem of the Arabidopsis Thaliana Inflorescence.

The regulation of sugar metabolism and partitioning plays an essential role for a plant's acclimation to its environment, with specific responses in autotrophic and heterotrophic organs. In this work, we analyzed the effects of high salinity on sugar partitioning and vascular anatomy within the floral stem. Stem sucrose and fructose content increased, while starch reduced, in contrast to the response observed in rosette leaves of the same plants. In the stem, the effects were associated with changes in the expression of SWEET and TMT2 genes encoding sugar transporters, SUSY1 encoding a sucrose synthase and several FRK encoding fructokinases. By contrast, the expression of SUC2, SWEET11 and SWEET12, encoding sugar transporters for phloem loading, remained unchanged in the stem. Both the anatomy of vascular tissues and the composition of xylem secondary cell walls were altered, suggesting that high salinity triggered major readjustments of sugar partitioning in this heterotrophic organ. There were changes in the composition of xylem cell walls, associated with the collapse and deformation of xylem vessels. The data are discussed regarding sugar partitioning and homeostasis of sugars in the vascular tissues of the stem.

Leaf wounding or simulated herbivory in young N. attenuata plants reduces carbon delivery to roots and root tips.

MAIN CONCLUSION: In Nicotiana attenuata seedlings, simulated herbivo ry by the specialist Manduca sexta decreases root growth and partitioning of recent photoassimilates to roots in contrast to increased partitioning reported for older plants. Root elongation rate in Nicotiana attenuata has been shown to decrease after leaf herbivory, despite reports of an increased proportion of recently mobilized photoassimilate being delivered towards the root system in many species after similar treatments. To study this apparent contradiction, we measured the distribution of recent photoassimilate within root tissues after wounding or simulated herbivory of N. attenuata leaves. We found no contradiction: herbivory reduced carbon delivery to root tips. However, the speed of phloem transport in both shoot and root, and the delivery of recently assimilated carbon to the entire root system, declined after wounding or simulated herbivory, in contrast with the often-reported increase in root partitioning. We conclude that the herbivory response in N. attenuata seedlings is to favor the shoot and not bunker carbon in the root system.

Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions.

Soil bacteria such as pseudomonads may reduce pathogen pressure for plants, both by activating plant defence mechanisms and by inhibiting pathogens directly due to the production of antibiotics. These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to plant health. A split-root system was set up with barley to quantify systemic and local effects of pre-inoculation with Pseudomonas fluorescens on the subsequent infection process by the fungal pathogen Fusarium graminearum. One root half was inoculated with F. graminearum in combination with P. fluorescens strain CHA0 or its isogenic antibiotic-deficient mutant CHA19. Bacteria were inoculated either together with the fungal pathogen or in separate halves of the root system to separate local and systemic effects. The short-term plant response to fungal infection was followed by using the short-lived isotopic tracer (11)CO(2) to track the delivery of recent photoassimilates to each root half. In the absence of bacteria, fungal infection diverted carbon from the shoot to healthy roots, rather than to infected roots, although the overall partitioning from the shoot to the entire root system was not modified. Both local and systemic pre-inoculation with P. fluorescens CHA0 prevented the diversion of carbon as well as preventing a reduction in plant biomass in response to F. graminearum infection, whereas the non-antibiotic-producing mutant CHA19 lacked this ability. The results suggest that the activation of plant defences is a central feature of biocontrol bacteria which may even surpass the effects of direct pathogen inhibition.

A Jasmonate-Induced Defense Elicitation in Mature Leaves Reduces Carbon Export and Alters Sink Priority in Grape (Vitis vinifera Chardonnay).

This work aims to understand how Vitis vinifera (Chardonnay) vines prioritise the export and distribution of recently fixed photoassimilate between root tissue, fruit, and defence, following the elicitation of a defence response. Jasmonic acid (JA) and its methyl ester, MeJA, are endogenous plant hormones, known collectively as jasmonates, that have signalling roles in plant defence and consequently are often used to prime plant defence systems. Here, we use exogenous jasmonate application to mature source leaves of Chardonnay grapevines to elucidate the prioritisation strategy of carbon allocation between plant defence and growth. Our results demonstrate that jasmonate application to Chardonnay leaves can elicit a defence response to Botrytis cinerea, but the effect was localised to the jasmonate-treated area. We found no evidence of a systemic defence response in non-treated mature leaves or young growing tissue. JA application reduced the photosynthetic rate of the treated leaf and reduced the export rate of recently fixed carbon-11 from the leaf. Following JA application, a greater proportion of available recently fixed carbon was allocated to the roots, suggesting an increase in sink strength of the roots. Relative sink strength of the berries did not change; however, an increase in berry sugar was observed seven days after JA treatment. We conclude that the data provide evidence for a "high sugar resistance" model in the mature treated leaves of the vine, since the export of carbon was reduced to ensure an elevated defence response in the treated leaf. The increase in berry sugar concentration seven days after treatment can be explained by the initial prioritisation of a greater portion of the exported carbon to storage in the roots, making it available for remobilisation to the berries once the challenge to defence had passed.

Root cooling strongly affects diel leaf growth dynamics, water and carbohydrate relations in Ricinus communis.

In laboratory and greenhouse experiments with potted plants, shoots and roots are exposed to temperature regimes throughout a 24 h (diel) cycle that can differ strongly from the regime under which these plants have evolved. In the field, roots are often exposed to lower temperatures than shoots. When the root-zone temperature in Ricinus communis was decreased below a threshold value, leaf growth occurred preferentially at night and was strongly inhibited during the day. Overall, leaf expansion, shoot biomass growth, root elongation and ramification decreased rapidly, carbon fluxes from shoot to root were diminished and carbohydrate contents of both root and shoot increased. Further, transpiration rate was not affected, yet hydrostatic tensions in shoot xylem increased. When root temperature was increased again, xylem tension reduced, leaf growth recovered rapidly, carbon fluxes from shoot to root increased, and carbohydrate pools were depleted. We hypothesize that the decreased uptake of water in cool roots diminishes the growth potential of the entire plant - especially diurnally, when the growing leaf loses water via transpiration. As a consequence, leaf growth and metabolite concentrations can vary enormously, depending on root-zone temperature and its heterogeneity inside pots.

Rapid cooling triggers forisome dispersion just before phloem transport stops.

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca(2+), and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re-condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour--a cooling rate-dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca(2+)-dependent sealing proteins that are present in all dicots.

Contrasting dynamics of water and mineral nutrients in stems shown by stable isotope tracers and cryo-SIMS.

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de-couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H(2)(18)O), magnesium ((26)Mg), potassium ((41)K) and calcium ((44)Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross-sections with cryo-secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross-section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up-regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.

In Vivo Veritas: Radiotracers in Studies of Phloem Transport of Carbohydrate.

Opportunities and challenges in the use of radiotracers to measure phloem transport are discussed, with an emphasis on noninvasive techniques to trace photoassimilate, carbon's short-lived isotope 11C, and an eye to pitfalls and traps to avoid. We discuss in turn the rationale for using tracers, the limitations and complications with using short-lived radiotracers like 11C, the physics of decay and detection that need to be known when data are interpreted, and methods of analysis.

Using Aphids to Measure Turgor Pressure Inside Sieve Elements.

The phloem is the long-distance transport system for photoassimilates within the plant. The vulnerability of the phloem tissue to blockage and damage makes it technically difficult to study, which has made it one of the least understood tissues. Transport of solution through the phloem appears to be by osmotically driven bulk flow, making an understanding of phloem hydrostatic pressure important in our comprehension of phloem flow mechanics. Here we describe a method of making in vivo direct transient measurements of phloem hydrostatic pressure using excised aphid stylets to directly access the phloem.

Arabidopsis Natural Accessions Display Adaptations in Inflorescence Growth and Vascular Anatomy to Withstand High Salinity during Reproductive Growth.

Plant responses to abiotic stresses entail adaptive processes that integrate both physiological and developmental cues. However, the adaptive traits that are involved in the responses to a high soil salinity during reproductive growth are still poorly studied. To identify new clues, we studied the halophyte, Thellungiella salsuginea, and three Arabidopsis accessions, known as tolerant or salt-sensitive. We focused on the quantitative traits associated with the stem growth, sugar content, and anatomy of the plants subjected to the salt treatment, with and without a three-day acclimation, applied during the reproductive stage. The stem growth of Thellungiella salsuginea was not affected by the salt stress. By contrast, salt affected all of the Arabidopsis accessions, with a natural variation in the effect of the salt on growth, sugar content, and stem anatomy. In response to the high salinity, irregular xylem vessels were observed, independently of the accession's tolerance to salt treatment, while the diameter of the largest xylem vessels was reduced in the tolerant accessions. The stem height, growth rate, hexoses-to-sucrose ratio, and phloem-to-xylem ratio also varied, in association with both the genotype and its tolerance to salt stress. Our findings indicate that several quantitative traits for salt tolerance are associated with the control of inflorescence growth and the adjustment of the phloem-to-xylem ratio.

Jasmonic acid treatment to part of the root system is consistent with simulated leaf herbivory, diverting recently assimilated carbon towards untreated roots within an hour.

It is known that shoot application of jasmonic acid (JA) leads to an increased carbon export from leaves to stem and roots, and that root treatment with JA inhibits root growth. Using the radioisotope (11)C, we measured JA effects on carbon partitioning in sterile, split-root, barley plants. JA applied to one root half reduced carbon partitioning to the JA-treated tissue within minutes, whereas the untreated side showed a corresponding--but slower--increase. This response was not observed when instead of applying JA, the sink strength of one root half was reduced by cooling it: there was no enhanced partitioning to the untreated roots. The slower response in the JA-untreated roots, and the difference between the effect of JA and temperature, suggest that root JA treatment caused transduction of a signal from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves to the untreated root tissue, as was indeed observed 10 min after the shoot application of JA. This supports the hypothesis that the response of some plant species to both leaf and root herbivores may be the diversion of resources to safer locations.

Modelling phloem transport within a pruned dwarf bean: a 2-source-3-sink system.

A mechanistic model of carbon partitioning, based on the Münch hypothesis of phloem transport and implemented with PIAF-Münch modelling platform (Lacointe and Minchin 2008), was tested for an architecture more complex than any tested previously. Using 11C to label photosynthate, responses in transport of photosynthate within a heavily pruned dwarf bean plant (Phaseolus vulgaris L.) to changes in source and sink activities were compared with model predictions. The observed treatment responses were successfully predicted. However, the observations could not be completely explained if the modelled stem contained only one phloem pathway: tracer from a labelled leaf was always detected in both shoot apex and root, whichever of the two leaves was labelled. This shows that bidirectional flow occurred within the stem, with solute moving simultaneously in both directions. Nevertheless, a model architecture with very little more complexity could incorporate such bidirectional flow. We concluded that the model could explain the observations, and that the PIAF-Münch model platform can be expected to describe partitioning in even more complex architectures.

Non-invasive approaches for phenotyping of enhanced performance traits in bean.

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene-environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure-function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Leaf photosynthetic and solar-tracking responses of mallow, Malva parviflora, to photon flux density.

Malva parviflora L. (mallow) is a species that occupies high-light habitats as a weedy invader in orchards and vineyards. Species of the Malvaceae are known to solar track and anecdotal evidence suggests this species may also. How M. parviflora responds physiologically to light in comparison with other species within the Malvaceae remains unknown. Tracking and photosynthetic responses to photon flux density (PFD) were evaluated on plants grown in greenhouse conditions. Tracking ability was assessed in the growth conditions and by exposing leaves to specific light intensities and measuring changes in the angle of the leaf plane. Light responses were also determined by photosynthesis and chlorophyll fluorescence. Leaves followed a heliotropic response which was highly PFD-dependent, with tracking rates increasing in a curvilinear pattern. Maximum tracking rates were up to 20 degrees h(-1) and saturated for light above 1,300 micromol (photons) m(-2) s(-1). This high-light saturation, both for tracking (much higher than the other species), and for photosynthesis, confirmed mallow as a high-light demanding species. Further, because there was no photoinhibition, the leaves could capture the potential of an increased carbon gain in higher irradiance by resorting to solar tracking. Modelling suggested the tracking response could increase the annual carbon gain by as much as 25% compared with leaves that do not track the sun. The various leaf attributes associated with solar tracking, therefore, help to account for the success of this species as a weed in many locations worldwide.

11C-imaging: methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled.

The long-distance transport and actions of the phytohormone methyl jasmonate (MeJA) were investigated by using the short-lived positron-emitting isotope 11C to label both MeJA and photoassimilate, and compare their transport properties in the same tobacco plants (Nicotiana tabacum L.). There was strong evidence that MeJA moves in both phloem and xylem pathways, because MeJA was exported from the labeled region of a mature leaf in the direction of phloem flow, but it also moved into other parts of the same leaf and other mature leaves against the direction of phloem flow. This suggests that MeJA enters the phloem and moves in sieve tube sap along with photoassimilate, but that vigorous exchange between phloem and xylem allows movement in xylem to regions which are sources of photoassimilate. This exchange may be enhanced by the volatility of MeJA, which moved readily between non-orthostichous vascular pathways, unlike reports for jasmonic acid (which is not volatile). The phloem loading of MeJA was found to be inhibited by parachloromercuribenzenesulfonic acid (PCMBS) (a thiol reagent known to inhibit membrane transporters), and by protonophores carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) suggesting proton co-transport. MeJA was found to promote both its own transport and that of recent photoassimilate within 60 min. Furthermore, we found that MeJA can counter the inhibitory effect of the uncoupling agent, CCCP, on sugar transport, suggesting that MeJA affects the plasma membrane proton gradient. We also found that MeJA's action may extend to the sucrose transporter, since MeJA countered the inhibitory effects of the sulfhydryl reagent, PCMBS, on the transport of photoassimilate.

Stimulating natural defenses in poplar clones (OP-367) increases plant metabolism of carbon tetrachloride.

Groundwater contamination by carbon tetrachloride (CCl4) presents a health risk as a potential carcinogen and pollutant that is capable of depleting the ozone layer. Although use of poplar trees in a phytoremediation capacity has proven to be cost effective for cleaning contaminated sites, minimizing leaf emission of volatile contaminants remains a pressing issue. We hypothesized that recently fixed carbon plays a key role in CCl4 metabolism in planta yielding nonvolatile trichloroacetic acid (TCA) and that the extent of this metabolism can be altered by heightening plant defenses. Labeling intact leaves with (11)CO2 (t 1/2 20.4 m) can test this hypothesis, because the extremely short half-life of the tracer reflects only those processes involving recently fixed carbon. Using radio-HPLC analysis, we observed [(11)C]TCA from leaf extract from poplar clones (OP-367) whose roots were exposed to a saturated solution of CCl4 (520 ppm). Autoradiography of [(11)C]photosynthate showed increased leaf export and partitioning to the apex within 24 h of CCl4 exposure, suggesting that changes in plant metabolism and partitioning of recently fixed carbon occur rapidly. Additionally, leaf CCl4 emissions were highest in the morning, when carbon pools are low, suggesting a link between contaminant metabolism and leaf carbon utilization. Further, treatment with methyljasmonate, a plant hormone implicated in defense signal transduction, reduced leaf CCl4 emissions two-fold due to the increased formation of TCA.

Phloem hydrostatic pressure relates to solute loading rate: a direct test of the Münch hypothesis.

According to the Münch hypothesis, a flow of solution through the sieve tubes is driven by a hydrostatic pressure difference between the source (or collection) phloem and the sink (or release) phloem. A high hydrostatic pressure is maintained in the collection phloem by the active uptake of sugar and other solutes, with a concomitant inflow of water. A lower pressure is maintained in the release phloem through solute unloading. In this work we directly test the role of solute uptake in creating the hydrostatic pressure associated with phloem flow. Solute loading into the phloem of mature leaves of barley and sow thistle was reduced by replacing the air supply with nitrogen gas. Hydrostatic pressure in adjacent sieve elements was measured with a sieve-element pressure probe, a cell pressure probe glued to the exuding stylet of aphids that had been feeding from the phloem. Sieve element sap was sampled by aphid stylectomy; sap osmotic pressure was determined by picolitre osmometry and its sugar concentration by enzyme-linked fluorescence assays. Samples were taken with a time resolution of ~2-3 min. In accordance with Münch's proposal a drop in osmotic and hydrostatic pressure in the source phloem following treatment of the source leaf with N2 was observed. A decrease in sugar concentration was the major contributor to the change in osmotic pressure. By observing these variables at a time resolution of minutes we have direct observation of the predictions of Münch.

Short-term storage of carbohydrate in stem tissue of apple (Malus domestica), a woody perennial: evidence for involvement of the apoplast.

This work investigates the pathway and mechanism for lateral retrieval of carbohydrate into the transport phloem of apple stems (Malus domestica Borkh.). A heat exchanger was set up on the stem, allowing rapid chilling and subsequent re-warming of stem segments while the time course of axial transport of 11C-labelled photoassimilate was measured at a position ∼65 mm downstream of the heat exchanger. Whenever axial transport was blocked by a sudden chill at the heat exchanger, transport 65 mm downstream from the blockage immediately slowed but did not stop, showing that there was retrieval of solutes into the pathway (buffering), within that 65 mm of stem, to help maintain the axial flow. Use of PCMBS, an inhibitor of sugar transporters, showed that the buffering included retrieval of sugar from the apoplast. We concluded that in apple, apoplastic sugar in stem tissue can buffer phloem transport during short-term changes in supply and demand for carbohydrates. Buffering was stronger when mobile reserves in the stem were higher, for example late in the photoperiod, or if carbohydrate demand in the terminal sink was increased. We also suggest that the concentration of sugars in the apoplast is a regulator of carbohydrate storage and re-mobilisation.