Physiological Sink Research Articles

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to <em>Plasmodiophora brassicae</em>

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Lactate in contemporary biology: a phoenix risen.

After a century, it's time to turn the page on understanding of lactate metabolism and appreciate that lactate shuttling is an important component of intermediary metabolism in vivo. Cell‐cell and intracellular lactate shuttles fulfil purposes of energy substrate production and distribution, as well as cell signalling under fully aerobic conditions. Recognition of lactate shuttling came first in studies of physical exercise where the roles of driver (producer) and recipient (consumer) cells and tissues were obvious. Moreover, the presence of lactate shuttling as part of postprandial glucose disposal and satiety signalling has been recognized. Mitochondrial respiration creates the physiological sink for lactate disposal in vivo. Repeated lactate exposure from regular exercise results in adaptive processes such as mitochondrial biogenesis and other healthful circulatory and neurological characteristics such as improved physical work capacity, metabolic flexibility, learning, and memory. The importance of lactate and lactate shuttling in healthful living is further emphasized when lactate signalling and shuttling are dysregulated as occurs in particular illnesses and injuries. Like a phoenix, lactate has risen to major importance in 21st century biology.

Clubroot Disease Stimulates Early Steps of Phloem Differentiation and Recruits SWEET Sucrose Transporters within Developing Galls.

Successful biotrophic plant pathogens can divert host nutrition toward infection sites. Here we describe how the protist Plasmodiophora brassicae establishes a long-term feeding relationship with its host by stimulating phloem differentiation and phloem-specific expression of sugar transporters within developing galls. Development of galls in infected Arabidopsis (Arabidopsis thaliana) plants is accompanied by stimulation of host BREVIS RADIX, COTYLEDON VASCULAR PATTERN, and OCTOPUS gene expression leading to an increase in phloem complexity. We characterized how the arrest of this developmental reprogramming influences both the host and the invading pathogen. Furthermore, we found that infection leads to phloem-specific accumulation of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS11 and 12 facilitating local distribution of sugars toward the pathogen. Utilizing Fourier-transform infrared microspectroscopy to monitor spatial distribution of carbohydrates, we found that infection leads to the formation of a strong physiological sink at the site of infection. High resolution metabolic and structural imaging of sucrose distributions revealed that sweet11 sweet12 double mutants are impaired in sugar transport toward the pathogen, delaying disease progression. This work highlights the importance of precise regulation of sugar partitioning for plant-pathogen interactions and the dependence of P brassicae's performance on its capacity to induce a phloem sink at the feeding site.

Insights into secondary growth in perennial plants: its unequal spatial and temporal dynamics in the apple (Malus domestica) is driven by architectural position and fruit load.

Secondary growth is a main physiological sink. However, the hierarchy between the processes which compete with secondary growth is still a matter of debate, especially on fruit trees where fruit weight dramatically increases with time. It was hypothesized that tree architecture, here mediated by branch age, is likely to have a major effect on the dynamics of secondary growth within a growing season. Three variables were monitored on 6-year-old 'Golden Delicious' apple trees from flowering time to harvest: primary shoot growth, fruit volume, and cross-section area of branch portions of consecutive ages. Analyses were done through an ANOVA-type analysis in a linear mixed model framework. Secondary growth exhibited three consecutive phases characterized by unequal relative area increment over the season. The age of the branch had the strongest effect, with the highest and lowest relative area increment for the current-year shoots and the trunk, respectively. The growth phase had a lower effect, with a shift of secondary growth through the season from leafy shoots towards older branch portions. Eventually, fruit load had an effect on secondary growth mainly after primary growth had ceased. The results support the idea that relationships between production of photosynthates and allocation depend on both primary growth and branch architectural position. Fruit load mainly interacted with secondary growth later in the season, especially on old branch portions.

Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

Ultrastructure of the calcium-sequestering gastrodermal cell in the hydroid Hydractinia symbiolongicarpus (Cnidaria, Hydrozoa).

Large, free-floating crystals of calcium carbonate occur in vacuoles of gastrodermal cells of the hydroid Hydractinia symbiolongicarpus. Here, morphological details about the process by which these cells accumulate and sequester calcium are provided by a cytochemical method designed to demonstrate calcium at the ultrastructural level. Electron-dense material presumably indicative of the presence of calcium was EGTA-sensitive and was shown by parallel electron energy loss spectroscopy (EELS) and energy spectroscopic imaging (ESI) to contain calcium. Calcium occurred in only one cell type, the endodermally derived gastrodermal cell. In these cells, the electron-dense material appeared first as a fine precipitate in the cytosol and nucleus and later as larger deposits and aggregates in the vacuole. During the life cycle, gastrodermal cells of the uninduced planula and the planula during metamorphic induction sequestered calcium. In primary polyps and polyps from established colonies, gastrodermal cells sequestered calcium, but the endodermal secretory cells did not. Our observations support the hypothesis that gastrodermal cells function as a physiological sink for calcium that enters the organism in conjunction with calcium-requiring processes such as motility, secretion, and metamorphosis.

Calcification in the planula and polyp of the hydroid Hydractinia symbiolongicarpus (Cnidaria, Hydrozoa).

This study examines calcification in planulae and polyps of the hydroid Hydractinia symbiolongicarpus. We observed that established colonies produce a crystalline mat on their substratum and that crystals visible by polarized light microscopy occur in the vacuoles of the gastrodermal cells of both polyps and planulae. The crystalline mat was found by infrared spectroscopy to contain calcium carbonate in the form of aragonite. The composition of the vacuolar crystals and the cellular mechanisms for manufacturing them were explored by alteration of calcium levels in the environment and by the use of pharmacological agents (acetazolamide, caffeine, DIDS, diltiazem, nifedipine, procaine, Ruthenium Red, ryanodine and verapamil) that affect cellular uptake and transport of calcium and bicarbonate. The results indicated that the crystals in the vacuoles contained calcium carbonate. The gastrodermal cells are hypothesized to serve as a physiological sink for excess calcium that enters the organism during motility, secretion and metamorphosis of the planula, and to create a crystalline substratum for the colony of polyps.

Ammonium release by nitrogen sufficient diatoms in response to rapid increases in irradiance

It has been hypothesized that nitrogen-replete diatoms, but not flagellates, may release NO2 - , NH4 + or dissolved organic nitrogen (DON) following rapid increases in irradiance (and conse- quently an increase in cellular electron energy), as might be expected to occur in a vertically well mixed estuarine system. Just as the increase in irradiance leads to an increase in cellular energy, so too would a decrease in temperature, due to the temperature dependency of biosynthetic enzymes. This hypothesis was tested by comparing the response of nitrogen-replete diatoms (Skeletomena costatum, Thalassiosira weissflogii and Chaetoceros sp.) and flagellates (Dunaliella tertiolecta, Pavlova lutheri and Prorocentrum minimum) to rapid increases in irradiance and decreases in temperature. Short-term (<3 h) changes in extracellular NO2 - and NH4 + concentrations were measured in cultures following these experimental shifts, as well as in cultures retained at the growth irradiance. Net rates of NO2 and NH4 + release were calculated from the time course of extracellular nitrogen concentra- tions. As a fraction of NO3 - uptake, NO2 - release rates under the increased irradiance increased marginally relative to NO2 - release rates under the growth irradiance. Release rates of NH4 + under the increased irradiance increased nearly fivefold over release rates at the growth irradiance, and accounted for 84% of the NO3 - uptake rate. In direct contrast to the diatom species, the flagellate species released NO2 under the higher experimental irradiance at rates one half those of the release rates under the growth irradiance, and continued to take up NH4 + under both irradiance conditions. Within the experimental boundaries, these findings have important physiological and ecological impli- cations. The magnitude of the observed nitrogen release represents a significant physiological sink for electrons and, in fact, calculations suggest that up to 62% of the total electrons harvested could be consumed. From an ecological perspective, these findings add to the body of literature which suggests that a significant fraction of the nitrogen that is taken up is ultimately released in dissolved form. More importantly, these data suggest that DON is not the only compound that phytoplankton may release in the aquatic environment.

Starch deposition in storage organs and the importance of nutrients and external factors

AbstractStarch is an important agricultural product deposited in vegetative and reproductive storage organs (sinks) of various crop species. Starch yield may in some cases be limited by photosynthesis, i.e. source‐limited. This is particularly true for starch synthesized in potato tubers. Here, the physiological sink is characterized by a symplastic phloem unloading path. In reproductive storage tissues (seeds), however, photosynthates must pass the apoplast on their path from phloem unloading to the storage cell. In cereal grains, phloem unloading of sucrose and poslunloading processes rather than photosynthesis may thus control starch synthesis (sink‐limited). Various limiting steps along the path of photosynthate movement from the phloem to the storage cells are considered.The primary organic carbon for starch synthesis is sucrose. Sucrose delivered to the storage cell is metabolized to UDPglucose and fructose by means of sucrose synthase activity. Concerning sucrose breakdown the role of cell‐wall bound invertase is not well defined. Competition for UDPglucose consumed for growth or storage may be a crucial process in photosynthate partitioning. High starch yields of crops require an undisturbed growth of the sink organ and an optimal filling of sink amyloplasts with starch. The most important form of organic carbon imported into amyloplasts of storage organs (cereal grain and potato tuber) and used for starch synthesis is glucose 1‐phosphate. It is still to be clarified whether the rate of glucose 1‐phosphate absorption has a direct impact on starch yield.In cereals, the total amount of starch accumulated depends significantly on the duration of grain filling. Ample nutrient and water supply at the post‐anthesis stage prolongs the period of grain filling and hence favours starch production. High temperature reduces the activity of soluble starch synthase with negative consequences for starch accumulation. The biochemical and physiological implications of these stress factors are discussed. Recently, successful transgenic manipulations of starch synthesis in crop plants have increased starch yield.

Subcellular Quantitative Determination of K and Ca in Phloem, Cambium, and Xylem Cells of Spruce(Picea abies[L.] Karst.) During Earlywood and Latewood Formation

The present investigation was directed to calibrated subcellular quantitative determination of K and Ca in phloem, cambium, and xylem cells of spruce (Picea abies [L.] Karst.) by energy dispersive X-ray spectroscopy (EDXS) in combination with transmission electron microscopy (TEM). The element content was studied during earlywood and latewood formation throughout pnmary and secondary wall formation and lignification. In the experimental stage, increment cores were extracted from three 100-year-old forest trees and either shock-frozen, embedded, and used for semithin cuttings, or prepared for optical emission spectroscopy (ICP-OES) for bulk analysis from tissue sections. The structural dynamics in cell formation of phloem, cambium, and xylem was expressed in terms of the radial cell diameter and the cell wall area. The lignification of the same cells was studied by subcellular UV-spectroscopy. During earlywood formation, differentiating tracheids are a strong physiological sink for K. Particularly during cell enlargement, a strong increase of the symplasmatic K content (> I ng per cell) was detected. An increase of the Ca content of differentiating cells (0.4-1 ng per cell) was found during secondary wall formation, whereas the Ca content of the cell wall decreased remarkably during its lignification (0.1-0.3 ng per cell). From these experiments it can be concluded that K is a driving force in cell enlargement of differentiating tracheids (primary wall phase), whereas Ca is of importance especially for wall synthesis in the secondary phase of xylem cell development and lignification.

Relative Growth Rate and its Relationship to Compositional Changes of Nonstructural Carbohydrates in the Mesocarp of Developing Peach Fruits

Dry weights of whole fruit and of different fruit tissues, such as the mesocarp (with exocarp) and the endocarp (with seed), were accumulated on early (`Spring Lady'), midseason (`Flamecrest'), and late-maturing (`Cal Red') peach [Prunus persica (L.) Batsch] cultivars during the 1988 growing season. Seasonal relative growth rate (RGR) patterns of whole fruit showed two distinct phases for `Flamecrest' and `Cal Red'; however, `Spring Lady' did not exhibit two distinct RGR phases. The shift from phase I to phase II of the whole fruit RGR curve was related to an intersection of mesocarp and endocarp RGR curves, indicating a change of physiological sink activities in those fruit tissues in the later-maturing cultivars, but not in the early cultivar. Nonstructural carbohydrate compositional changes in concentration or content were similar in the three peach cultivars. Sucrose accounted for most of the seasonal increase in mesocarp nonstructural carbohydrate concentration. A sudden rise of sucrose was associated with the phase shift of the fruit RGR curves of the midseason and late-maturing cultivars, but not of the early maturing cultivar; however, in the early maturing cultivar, mesocarp compositional carbohydrate changes and, particularly, the sucrose increase, indicate that the physiological processes normally associated with the two phases exist in very early maturing fruit but are not associated distinctly with two separate RGR phases.

The development of endomycorrhizal root systems

SUMMARYConcentrations of phosphorus in shoot and soluble carbohydrates (fructose, glucose, sucrose and fructans) in root were measured in non‐mycorrhizal and vesicular‐arbuscular (VA) mycorrhizal (Glomus mosseae) leek plants (Allium porrum) raised at six concentrations of soil phosphate. In conditions when an increased concentration of soil phosphate reduced VA mycorrhizal infection, the concentrations of soluble carbohydrates in the root were at a maximum. Therefore the hypothesis that greater concentrations of soluble carbohydrates in roots favour VA mycorrhizal infection is discounted. There was a specific effect of VA mycorrhizas, in that infected roots contained a larger concentration of sucrose than did uninfected roots, in plants with similar phosphorus concentrations in dry matter of shoots.We conclude, first, that increased phosphorus supply from either phosphate addition to soil or VA mycorrhizal infection increases concentration of soluble carbohydrates in leek roots and, secondly, that the VA mycorrhizal root behaves as a particularly strong physiological sink when there is an excess concentration of sucrose in the host.

Red Clover Mottle Virus Infection Affects Sink-Source Relationships and Starch Accumulation in Pea Plants

It has frequently been observed that starch accumulates in plant tissues after virus infection. In pea plants infected with red clover mottle comovirus, strain «O», virus replicates in the inoculated leaves and at the shoot apex where it induces lethal top necrosis. Concomitant with the onset of top necrosis, starch accumulates in the intervening leaves which remain substantially virus-free. This pattern of starch accumulation can be mimicked by removal of the apex in uninfected plants. We conclude that in this plant-virus interaction starch accumulation is an indirect consequence of virus infection associated with the removal of the physiological sink for photosynthate

Absorption and Translocation of Glyphosate by Carrot Infected by Swamp Dodder

Absorption of “C-labeled glyphosate by whole carrot (Daucus carota L.) plants infected or not infected by swamp dodder (Cuscuta gronovii Willd ex R &amp; S) increased from 14% 1 day after treatment to 56% 14 days after treatment. Absorption of 14C-labeled glyphosate did not increase from 14 to 45 days after treatment. 14Carbon-labeled glyphosate appeared in the carrot root 1 day after application and its concentration increased with time in both infected and noninfected plants until 14 days after treatment. From 14 to 45 days after treatment, the concentration of 14C-labeled glyphosate in the roots decreased. At 1 day after treatment, dodder tissue contained as much 14C-labeled glyphosate as any physiological sink in the carrot. At 45 days after treatment, dodder tissue contained more 14C-labeled glyphosate than all other physiological sinks, except the petiole of the treated leaf. Swamp dodder stems had absorbed 14C-labeled glyphosate directly from a solution within 1 day after treatment. Chemical name used: N-(phosphonomethyl)glycine (glyphosphate).

Accumulation of copper and nickel in plant tissues and an insect gall of lowbush blueberry, Vaccinium angustifolium, near an ore smelter at Sudbury, Ontario, Canada

The accumulation of copper and nickel in plant tissues and galls of Hemadas nubilipennis on lowbush blueberry near an ore smelter at Sudbury, Ontario, was investigated. Concentrations of these metals in the root, stem, and leaf tissue decline logarithmically with increasing distance from the Sudbury smelter. The pattern of accumulation for copper and nickel in the various tissues was root &gt; stem &gt; leaf &gt; berry; however, metal differences in these tissues were not as great in plants farther from the smelter. The root tissue is the primary site of accumulation of these metals when environmental levels of copper and nickel are high. The highest concentrations of copper and nickel were found in the galls, indicating that gall tissues act as a strong physiological sink for micronutrients and redirect nutrients from the host plant. Key words: Vaccinium angustifolium, copper, nickel, gall, Sudbury.

Effect of Potassium on the Assimilate Conduction to Storage Tissue

AbstractThe promoting effect of K+ on phloem transport has been shown by various authors for different plant species. This effect was also obtained in cases in which C02‐assimilation was not improved by K+. Experiments with Ricinus communis provided evidence that K+ favoured the flux rate of phloem sap without diluting its content in organic and inorganic solutes. This all over effect results in higher flux rates of all phloem solutes. In this respect the long distance transport of sucrose, amino acids and Mg2+ is of particular interest. The long distance transport of these compounds was shown to be favoured by K+.Potassium is known to activate starch and protein synthezising enzymes involved in the metabolism of storage tissue. There is evidence, that also in cases of a suboptimum K+ supply, the K+ level in the storage tissue probably is still high enough to provide an optimum activation of the K+ requiring enzymes. For this reason it is unlikely that the favourable effect of K+ on phloem transport is related to processes in the physiological sink. It is more likely that K+ favours the phloem loading process. Indirectly K+ could promote the phloem loading process by improving the provision of energy (ATP). It is, however, also feasible that K+ acts more directly by being involved in the ATPase reaction which is supposed to drive the sucrose uptake of sieve tubes.

Amylase and Invertase Activities and Carbohydrate Contents in Relation to Physiological Sink in Carnation

AbstractIn carnation, Dianthus caryophyllus L. cv. Hanacy, initiation of flower primordia was followed by mobilization of polysaccharides from leaves to developing flower buds and mature flowers. This process functioned by integrative activities of amylase and invertase, ensuring abundant flow of the mobilized carbohydrates from the source to the sink. Successive stages of floral development exhibited different sink strengths as indicated by amylase and invertase activities and carbohydrate contents in stem apices, which increased with progression of floral development.

Amylase and invertase activities in relation to the concept of a ‘physiological sink’ in potato plants grown in different seasons, and the influence of chlormequat upon these

Field trials were done to study the relation of dry matter accumulation, tuberization, and total carbohydrate content in different plant organs with the activities of amylase and invertase in different growth seasons (winter and summer). The activities of both enzymes showed higher levels in winter and were correlated with lower total carbohydrate contents in leaves. However, dry matter production in tubers was higher in winter. It is suggested that higher hydrolytic activity might therefore be needed in winter for the mobilization of the stored carbohydrates in the leaves for transport to the physiological sink. In the conditions of earlier tuberisation and more dry matter production, higher activities of amylase and invertase were found. Chlormequat treatments led to higher amylase activity in leaves correlated with earlier tuberisation and higher tuber yield, thouth invertase activity was not affected or rather slightly decreased.

MORPHOGENETIC ASPECTS OF A LEAFLESS MUTANT IN TOMATO II. INDUCTION OF A VASCULAR CAMBIUM

The vascular tissues of the leafless form of the “lanceolate” mutant in tomato resemble those of the normal hypocotyl in the lower portions. A divergence in development occurs in the middle region of the mutant hypocotyl: two xylary strands split acropetally into five to seven units, while in the normal hypocotyl four main vascular bundles are established. The three to four week old mutant hypocotyl shows a marked proliferation of cells in its central cylinder when it is grown on a simple nutrient medium; proliferation also occurs in older specimens grown in soil. This condition is averted if the mutant is grafted beneath an actively growing normal shoot tip. Instead of having numerous, random cell divisions in the central cylinder, the grafted mutant shows large pith parenchyma cells and an active vascular cambium. These features characterize the normal hypocotyl and they have not been observed in the intact leafless mutant. The observed behavior of intact and grafted mutant hypocotyls can be interpreted in terms of the normal shoot tip acting both as physiological sink, with respect to cell‐division stimuli produced mainly in the root system, and physiological source, with respect to cambial activating hormones.