Phloem Translocation and Heat-induced Callose Formation in Field-grown Gossypium hirsutum L

Phloem translocation rates in field-grown cotton (Gossypium hirsutum L.) dropped from morning to afternoon and continued to decline toward evening, except that recovery occurred following the hottest afternoon when the maximum temperature was 44 C. Water deficits increased from morning to evening, and severity of deficits generally were proportional to daytime heating. Water stress contributed toward reducing translocation but was not always the governing factor. Callose breakdown appeared to be slower than heat-induced synthesis, and in the evening callose still reflected the influence of high afternoon temperatures. Translocation was considerably reduced when about 50% or more of the hypocotyl sieve plates had large amounts of callose. While heat-induced callose may have reduced translocation because of sieve plate pore constriction, temperatures of 39 to 44 C appeared to inhibit an additional component of translocation as well, possibly in the leaf blade.     

An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation

Arabidopsis was transformed with double-stranded RNA interference (dsRNAi) constructs designed to silence three putative callose synthase genes: GLUCAN SYNTHASE-LIKE5 (GSL5), GSL6, and GSL11. Both wound callose and papillary callose were absent in lines transformed with GSL5 dsRNAi and in a corresponding sequence-indexed GSL5 T-DNA insertion line but were unaffected in GSL6 and GSL11 dsRNAi lines. These data provide strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation. Deposition of callosic plugs, or papillae, at sites of fungal penetration is a widely recognized early response of host plants to microbial attack and has been implicated in impeding entry of the fungus. Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis. Paradoxically, the absence of callose in papillae or haustorial complexes correlated with the effective growth cessation of several normally virulent powdery mildew species and of Peronospora parasitica.     

Ultrastructural Observations on CTC-Induced Callose Formation in Riella helicophylla

The Ca²⁺-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after 10 min, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone. At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeCl₃, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.     

The Effect of Inhibitors on Callose Formation in the Sieve Tubes of Saxifraga sarmentosa L.

Electron-microscope studies on stolons undergoing transportinhibition under nitrogen, HCN, and DNP treatment show no evidenceof callose-blockage of the sieve plates. Inhibition must thereforebe interpreted in terms of interruption of the sieve-tube energysupply; this weights the evidence in favour of theories of activemass flow such as those invoking electro-osmosis or proteincontractility.     

Rhizobium-stimulated Callose Formation in Clover Root Hairs and its Relation to Infection

Callose was detected in the cell walls of the tips of growingroot hairs of Trifolium species and the non-legume Phleum pratenseusing u.v. fluorescence of fresh material stained with 0·005%aniline blue. Inoculation of the roots with Rhizobium trifolii,R. leguminosarum, R. meliloti, and R. japonicum, or additionof 10^–7 and 10^–8 M indole-3-acetic acid (IAA) increasedtip callose formation. Most tip callose was formed at 12 °C, and amounts declinedprogressively at 18, 24, and 30 °C, with very little formedat 36 °C. Tip calloso usually became less and disappearedin individual root hairs as they aged. Callose which appeared prominently in the host cell walls atthe points of initiation of infection threads did not usuallydisappear as the hairs matured. There was little or no extensionof callose along the infection thread and none in the threadtip or in the cell nucleus. Presumptive regions of callose hadsimilar structure and electron density as root hair wall materialand were sometimes related to arrays of vesicles in the hostcytoplasm. The external surface of the hair wall bore smallpegs or papillae (0·1–0·2 µm) continuouswith the outer layer of the wall and possibly associated withattachment of bacteria. Bacteria were usually umboriate at thepoint of attachment and their polyphosphate granules were muchlarger near the root hair than at the distal end.     

Preliminary Experiments on the Effect of Temperature on the Movement of 14C-labelled Assimilates through the Phloem of Willow

Experiments were performed on both young shoots of willow (3–5weeks old) and mature stems which had been growing for a periodof 2–3 years. ¹⁴CO₂ was supplied to the leaves, and themass transport of the labelled assimilates through a portionof the stem enclosed in a temperature-controlled jacket wasmeasured by determining the slope of the rise in the activityof huneydew collected from a colony of the aphid Tuberolachnussalignus (Gmelin). It was found that there was a marked difference in the behaviourof the young shoots and mature stems. In the former, a fallin temperature slowed the transport, whilst in the latter acomparable fall in temperature increased the transport. Possiblecauses for these effects have been suggested, and the resultsare discussed in relation to published work on temperature effects.     

Callose synthesis in higher plants

Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches and it exists in the cell walls of a wide variety of higher plants. Callose plays important roles during a variety of processes in plant development and/or in response to multiple biotic and abiotic stresses. It is now generally believed that callose is produced by callose synthases and that it is degraded by β-1,3-glucanases. Despite the importance of callose in plants, we have only recently begun to elucidate the molecular mechanism of its synthesis. Molecular and genetic studies in Arabidopsis have identified a set of genes that are involved in the biosynthesis and degradation of callose. In this mini-review, we highlight recent progress in understanding callose biosynthesis and degradation and discuss the future challenges of unraveling the mechanism(s) by which callose synthase operate.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, pollen, plasmodesmata, cell plate, stress     

Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.A series of elegant experiments have demonstrated recently that phloem samples collected from cut petioles and stems of cucurbits do not represent pure fascicular phloem sap but rather the mixed content of extrafascicular phloem (EFP), xylem, and fascicular phloem (Zhang et al., 2010, 2012). The EFP is a unique feature of Cucurbitaceae. It consists of a complex network of longitudinal perifascicular strands next to the fascicular bundles, lateral commissural strands, and entocyclic as well as ectocyclic sieve tubes (Zhang et al., 2012). In contrast to the fascicular phloem, the EFP does not build effective callose plugs and freely exudes from cut sieve tubes. Due to easy sampling and its high protein content, cucurbit exudates were frequently used for phloem biochemistry (van Bel and Gaupels, 2004; Turgeon and Oparka, 2010; Atkins et al., 2011). Recently, more than 1,100 phloem proteins were identified in a large-scale proteomic approach with pumpkin (Cucurbita maxima; Lin et al., 2009). Interestingly, 67%, 46%, and 62% of the previously identified phloem proteins from rice (Oryza sativa), rape (Brassica napus), and castor bean (Ricinus communis), respectively, were found among the EFP proteins of pumpkin, confirming functional overlap between extrafascicular and fascicular phloem of different plant species (Lin et al., 2009).Although the EFP is physically and functionally linked to the fascicular phloem, a role in assimilate transport, the major function of fascicular phloem, is still ambiguous. The presence of many defense-related proteins in cucurbit phloem exudates rather pointed toward a role of the EFP in (systemic) stress and defense responses (van Bel and Gaupels, 2004; Walz et al., 2004; Turgeon and Oparka, 2010). In this regard, it has been largely overlooked by phloem biologists that phloem exudates of cucurbits are routinely classified by ecologists as latex-like exudates involved in defense against herbivorous insects (Carroll and Hoffman, 1980; Tallamy, 1985; Konno, 2011). In fact, the EFP is similar to branched laticifer (latex-containing conduits) networks, which develop from protophloem and/or phloem initials (Hagel et al., 2008). For this reason, and for better differentiation from fascicular phloem samples, hereafter we will use the term phloem latex instead of phloem exudates.Phloem latex provides two layers of defense. It is a physical barrier for small insects, which can be trapped in large droplets of exudates from wounded veins or sticky compounds that might glue their mouth parts (Konno, 2011). In addition, it was also shown that compounds in phloem latex of squash (Cucurbita spp.) such as cucurbitacin steroids deterred beetles from feeding (Carroll and Hoffman, 1980; Tallamy, 1985). Specialist feeders of cucurbits can tolerate toxic compounds in phloem latex or even use them for their own defense. Other herbivores, such as certain species from the genus Epilachna, counteract chemical defense by trenching. They isolate a circular leaf area by cutting all tissues except for the lower epidermis, this way avoiding pressure-driven exudation within the feeding area (Carroll and Hoffman, 1980; Tallamy, 1985; Konno, 2011).Some defense responses were demonstrated to be inducible by herbivore attack both in the local as well as neighbor leaves (Carroll and Hoffman, 1980; Tallamy, 1985). In this report, we analyzed by gas chromatography/mass spectrometry (GC-MS) and stable isotope-coded protein labeling (ICPL) systemic wound responses of the EFP upon leaf wounding. Overall, wounding induced jasmonate accumulation, reprogramming of the metabolism toward increased energy status, and the regulation of proteins related to carbon metabolism, signaling, and defense. This report gives a comprehensive overview of wound-inducible changes in the metabolite and the protein composition of pumpkin phloem latex, thereby providing a framework for future in-depth studies on defense responses of both EFP as well as fascicular phloem.     

The Electro-osmotic Theory of Phloem Transport in the Light of Recent Measurements on Heracleum Phloem

The bearing, on the electro-osmotic theory, of work on the Onsagercoefficients for Heracleum phloem, currently reported by Tyreeand Fensom (1970), is briefly discussed. As an aid to the discussionthe Onsager resistance coefficients are evaluated in terms ofSpiegler's frictional model. It is concluded that the experimentalevidence, while it does not support the theory, does not invalidateit. Some further suggestions regarding the electro-osmotic theoryare put forward.     

Reduced Population Control of an Insect Pest in Managed Willow Monocultures

Background

There is a general belief that insect outbreak risk is higher in plant monocultures than in natural and more diverse habitats, although empirical studies investigating this relationship are lacking. In this study, using density data collected over seven years at 40 study sites, we compare the temporal population variability of the leaf beetle Phratora vulgatissima between willow plantations and natural willow habitats.

Methodology/Principal Findings

The study was conducted in 1999–2005. The density of adult P. vulgatissima was estimated in the spring every year by a knock-down sampling technique. We used two measures of population variability, CV and PV, to compare temporal variations in leaf beetle density between plantation and natural habitat. Relationships between density and variability were also analyzed to discern potential underlying processes behind stability in the two systems. The results showed that the leaf beetle P. vulgatissima had a greater temporal population variability and outbreak risk in willow plantations than in natural willow habitats. We hypothesize that the greater population stability observed in the natural habitat was due to two separate processes operating at different levels of beetle density. First, stable low population equilibrium can be achieved by the relatively high density of generalist predators observed in natural stands. Second, stable equilibrium can also be imposed at higher beetle density due to competition, which occurs through depletion of resources (plant foliage) in the natural habitat. In willow plantations, competition is reduced mainly because plants grow close enough for beetle larvae to move to another plant when foliage is consumed.

Conclusion/Significance

To our knowledge, this is the first empirical study confirming that insect pest outbreak risk is higher in monocultures. The study suggests that comparative studies of insect population dynamics in different habitats may improve our ability to predict insect pest outbreaks and could facilitate the development of sustainable pest control in managed systems.     

The Breeding of the Willow Warbler

Climate is in a continual state of flux and, directly and indirectly, its fluctuations affect the status of plants, insects, birds and mammals (not excepting man). In this lecture Professor Lamb discusses the relationship between recent climatic shifts and the global atmospheric circulation, and raises the question of whether or not climatic forecasting is feasible.     

The Electroshock-Induced Inhibition of Phloem Translocation

The transient inhibition of phloem translocation which is producedby an electric shock was studied by applying controlled-currentstimuli to short lengths of bean stem. Translocation was monitoredby observing the accumulation of carbon-11 label into the plantapex. The principal findings are: (i) For constant-current electricshocks whose (current) x (time) product was held constant at10 mC, those of higher current (and shorter duration) gave longerlasting inhibitions, (ii) Breaking a 5 s pulse into a trainof 100 ms pulses slightly shortened the duration of the inhibition,even though the same total charge was passed; however, the separationof the pulses within the train did not seem to matter. But,when the inhibition produced by a pair of 50 ms pulses was studied,the duration of inhibition increased with pulse spacing. (iii)Single pulses as short as 10 ms could produce a detectable inhibition.The duration of inhibition grew rapidly with pulse length forpulse lengths between 10 ms and 100 ms. (iv) When a pair of2.5 s pulses was applied, a longer inhibition was obtained ifthe two pulses were of opposite polarity. To explain these phenomena,a qualitative conceptual model is provided in terms of ion channelsin the phloem. In the experiments reported here and in all previous pulse-labellingexperiments from this laboratory, it has been noted that rapidlyincreasing stochastic variability in the count data made itextremely difficult to obtain useful results for a period ofmore than 2 h after loading, even if the number of counts perunit time remained fairly high. A quantitative theory for thislimitation is worked out and shown to agree with experimentaldata. Therefore, since biological variability from plant toplant makes it advisable to compare matched stimuli on the sameplant in order to detect trends reliably, the number of challengeswhich can usefully be applied following a single pulse-labellingwith a short-lived isotope is quite limited. Key words: Phloem translocation, transient inhibition, electric shock     

The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems

The hypothesis that auxin (IAA) and gibberellic acid (GA₃) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA₃ (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA₃/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA₃ induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA₃/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.     

The evidence for symplastic Phloem loading

Experimental protocols used to study the route(s) of phloem loading in source leaves are evaluated; they include the analysis of plasmodesmatal ultrastructure and distribution, mobile compounds in the free space, loading patterns of exogenous sugars, the site of sugar synthesis, and dye coupling. All these methods have drawbacks. There are indications that some plants, especially those that translocate the raffinose series of sugars, load by a symplastic (through plasmodesmata) pathway, but the evidence is weak.