Ethylene and not embolism is required for wound-induced tylose development in stems of grapevines

The pruning of actively growing grapevines (Vitis vinifera) resulted in xylem vessel embolisms and a stimulation of tylose formation in the vessels below the pruning wound. Pruning was also followed by a 10-fold increase in the concentration of ethylene at the cut surface. When the pruning cut was made under water and maintained in water, embolisms were prevented, but there was no reduction in the formation of tyloses or the accumulation of ethylene. Treatment of the stems with inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine) and/or action (silver thiosulfate) delayed and greatly reduced the formation of tyloses in xylem tissue and the size and number of those that formed in individual vessels. Our data are consistent with the hypotheses that wound ethylene production is the cause of tylose formation and that embolisms in vessels are not directly required for wound-induced tylosis in pruned grapevines. The possible role of ethylene in the formation of tyloses in response to other stresses and during development, maturation, and senescence is discussed.     

Cu2+ inhibition of gel secretion in the xylem and its potential implications for water uptake of cut Acacia holosericea stems

Maintaining a high rate of water uptake is crucial for maximum longevity of cut stems. Physiological gel/tylosis formation decreases water transport efficiency in the xylem. The primary mechanism of action for post‐harvest Cu²⁺ treatments in improving cut flower and foliage longevity has been elusive. The effect of Cu²⁺ on wound‐induced xylem vessel occlusion was investigated for Acacia holosericea A. Cunn. ex G. Don. Experiments were conducted using a Cu²⁺ pulse (5 h, 2.2 mM) and a Cu²⁺ vase solution (0.5 mM) vs a deionized water (DIW) control. Development of xylem blockage in the stem‐end region 10 mm proximal to the wounded stem surface was examined over 21 days by light and transmission electron microscopy. Xylem vessels of stems stood into DIW were occluded with gels secreted into vessel lumens via pits from surrounding axial parenchyma cells. Gel secretion was initiated within 1–2 days post‐wounding and gels were detected in the xylem from day 3. In contrast, Cu²⁺ treatments disrupted the surrounding parenchyma cells, thereby inhibiting gel secretion and maintaining the vessel lumens devoid of occlusions. The Cu²⁺ treatments significantly improved water uptake by the cut stems as compared to the control.     

Comparison of petal senescence in forced and unforced common lilac flowers during their postharvest life

Recently, the programmed cell death (PCD) is studied in the context of the postharvest longevity of cut flowers with the goal of slowing down the processes that ultimately lead to flower death, and to ensure a long display life of cut plant material. In this study, the phenomenon of PCD in petals of common lilac (Syringa vulgaris L.) was observed, aimed to compare degradation of petal cells in flowers blooming under natural conditions with those forced in November. For the early lilac forcing, a deep dormancy has to be broken by high temperatures 35–37 °C negatively affecting postharvest life of cut branches. The trials included as well the observation of the effect of two flower preservatives on the PCD in order to see if the prolongation of the lilac vase life was associated with a delay in the onset of the PCD symptoms. The vase life of cut lilacs was significantly increased by both preservatives. The first symptoms of PCD were evident in the flower bud stage. In petals from forced shrubs, the first symptoms of cell degradation were much more advanced than in lilacs blooming naturally in May. In forced flowers held in the preservatives, the degradative changes in cells occurred later than in those kept in water, but they were accelerated relative to a flower developmental stage.     

Pruning-induced tylose development in stems of current-year shoots of Vitis vinifera (Vitaceae)

Tyloses form in xylem vessels in response to various environmental stimuli, but little is known of the kinetics or regulation of their development. Preliminary investigations indicated that wounds seal quickly with tyloses after pruning of grapevine shoots. In this study, tylose development was analyzed qualitatively and quantitatively at different depths and times from pruning cuts along current-year shoots of grapevines at basal, middle, and apical stem regions. Tyloses developed simultaneously within a single vessel but much separated in time among vessels. Pruning caused prodigious tylosis in vessels of grape stems, extending to approximately 1 cm deep and to 7 d after wounding, but about half of the vessels did not become completely occluded. The fraction of vessels forming tyloses was greatest in basal (85%) and least in apical (50%) regions. The depth of maximum density of tyloses was 4 mm from the cut in the basal region and 2 mm from the cut in the middle and apical regions. Tylose development was faster in the basal and middle than in the apical region. The pattern of tylose development is discussed in the context of wound repair and pathogen movement in grapevines.     

Development of tyloses in the xylem vessels of Meili grapevine and their effect on water transportation

Shoot pruning could cause short-term damages to vines. In response to damage, tyloses develop in shoot xylem vessels interfering free water and mineral transportation. In this study, the tylosis development at different nodes of the current-year and perennial shoots of sixty three-year-old grapevines (Vitis vinifera L.) after pruning was investigated. The results showed that tyloses at the nodes closest to the trunk developed rapidly; and tylosis development initiated at the time when the size of vessel-ray pit was greater than that of parenchyma cell-parenchyma cell pit. In current-year shoots, tyloses were formed in up to 87% of the vessels, and 40% of the vessels were completely blocked by tyloses. In wound-induced perennial shoots, 30% of the vessels were completely blocked by tyloses. When few vessels were blocked by tyloses, new vessels could differentiate, and water transportation system may be restored. However, when tyloses developed in a large number of vessels and the large number of the vessels were blocked, the original capability of water transport was decreased (the largest decrease was 21.1% in this study), resulting in dehiscence or shrinkage cracking in this area. The study proved that the tylosis formation in functional vessels limited the water transport efficiency.     

Wound-induced vascular occlusions in Vitis vinifera (Vitaceae): Tyloses in summer and gels in winter1

Vascular occlusion in xylem conduits is a common response to environmental stresses, and plant species are recognized as primarily tylose-forming or gel-forming. These stresses occur throughout the year, but there is little information on the wound responses throughout the year and in growing and dormant tissues. Wound-induced vascular occlusions were evaluated by type (tylose or gel), temporal progress, and spatial distribution for grape stems pruned in four seasons through an entire year. Tyloses were formed predominantly in summer and gels in winter. Cytohistological analyses indicated that wound-induced gels were pectin-rich. Both gel formation and tylose development were complete within 7 d and 10 mm from the cut regardless of the season of the wounding. Most vessels were affected by wounding, but a higher fraction of vessels developed occlusions in summer and autumn (over 80%) than in winter and spring (about 60%). The study is the first to show a single species is capable of producing primarily either tyloses or gels and that the type of wound-induced occlusion is dependent upon the season in which wounding occurs. Winter conditions limit the wound response to reversible gel formation that may contribute to refilling of embolized vessels in the spring.     

Cutting stems before relaxing xylem tension induces artefacts in Vitis coignetiae,as evidenced by magnetic resonance imaging

It was recently reported that cutting artefacts occur in some species when branches under tension are cut, even under water. We used non‐destructive magnetic resonance imaging (MRI) to investigate the change in xylem water distribution at the cellular level in Vitis coignetiae standing stems before and after relaxing tension. Less than 3% of vessels were cavitated when stems under tension were cut under water at a position shorter than the maximum vessel length (MVL) from the MRI point, in three of four plants. The vessel contents remained at their original status, and cutting artefact vessel cavitation declined to <1% when stems were cut at a position farther than the MVL from the MRI point. Water infiltration into the originally cavitated vessels after cutting the stem, i.e. vessel refilling, was found in <1% of vessels independent of cutting position on three of nine plants. The results indicate that both vessel cavitation and refilling occur in xylem tissue under tension following stem cutting, but its frequency is quite small, and artefacts can be minimized altogether if the distance between the monitoring position and the cutting point is longer than the MVL.     

Tyloses and the Maintenance of Transpiration

During a study of transpiration and embolism-formation in petiolesof sunflower, tyloses were frequently observed in early metaxylemvessels. Tyloses were confined to the inner ends of the xylemarcs, remote from the phloem. Vessels in this position are especiallyvulnerable to embolism. All stages of the invasion of vessellumens by xylem parenchyma cells were observed, from the earlyprotuberance of a cell through a pit to the complete occlusionof the lumen by one to several cells. The lumen space not occupiedby tyloses was seen both filled with xylem sap, or embolizedand gas-filled. Thus, during the early stages of tylosis formationthe vessel remained active in carrying the transpiration stream.Thin-walled vessels of the protoxylem or early metaxylem werenot tylosed, but were squashed and disappeared. These observationsare interpreted as evidence that vessels vulnerable to embolismare decommissioned and replaced by parenchyma tissue, whilenew and less vulnerable vessels are added to the xylem arcsat the cambial side. It is proposed that tylosis formation istriggered by the frequent embolization of the vulnerable vesselsto give, ultimately, an incompressible tissue. Then tyloseswould be necessary to preserve the tissue pressure which expresseswater to refill embolisms in the remaining vessels, and maintaintranspiration, as explained by the compensating pressure theoryof water transport. Compensating pressure theory; embolisms; starch sheath; tissue pressure; transpiration; tyloses; vessel diameter     

The effects of microbial exopolysaccharides (EPS) in vase water on the water relations and the vase life of Rosa cv. Sonia

Pure cultures of five microbial species were used to test the formation of exopolysaccharides (EPS) when grown in agitated sucrose (5% w/v) containing liquid cultures. These test species were isolated from stems of freshly harvested cut flowers ( Chrysanthemum, Gerbera and Rosa ) or from the vase water of these flower cultivars. The partial conversion of sucrose into other saccharides was demonstrated by HPLC and colorimetric analysis. The final polymeric character of the newly formed saccharides was investigated. SEM preparations of xylem vessels of Rosa maintained in EPS-containing vase water showed blockage, disorganization and injury of the vessel structure. EPS were shown not to pass the xylem pit membranes. Recovery from the first symptoms of disturbed water flow (wilting) due to EPS was possible in young flowers by cutting off the blocked part of the stem (15–20 cm. The higher the microbial conversion rate of sucrose into polysaccharides, the more disturbed were the water relations of the roses placed in the EPS-containing fluid, as was demonstrated by the decrease of: (1) water conductivity of Rosa stem segments (ml/30 min); (2) water uptake (ml/d); (3) Rosa vase life (d); and (4) flower bud development. Bacterial EPS (presumably levans and dextrans) could be concentrated in the retentate by molecular filtration with a cut-off level of 10000 Da. Filtrates did not cause Rosa xylem blockage and 'bent-neck'of the flower stems, but still may be toxic to roses. Two simple methods were also used for diagnostic investigations: (1) the beetroot tissue cube test to detect microbial products causing injury of the plant cell membranes, (2) the acid fuchsin test, to show the extent and location of Rosa xylem vessel occlusion.     

Micro-organisms at the cut surface and in xylem vessels of rose stems

Stems of cut rose flowers ( Rosa hybrida L. cv. 'Sonia') were placed in water to study the development of a population of micro-organisms at the cut surface and in the xylem vessels. The cut surface became covered with bacteria within 2 d of vase life. The bacteria were accompanied by an amorphous substance which was apparently bacterial slime. After 7 d of vase life many fungal hyphae were also found at the cut surface. Inside the xylem vessels the bacteria were often clustered at the inter-vessel pits. After 4 d of vase life most of the vessels that had been opened by cutting contained bacteria. Only a few xylem elements, located several centimeters from the cut surface, contained an amorphous substance. A few fungal hyphae were observed inside the xylem vessels. No yeasts were found, either at the cut surface or inside the xylem. Pseudomonas species accounted for more than 70% of the total bacterial population of the cut surface and the xylem vessels, and Enterobacter species (mainly Ent. agglomerans ) for less than 10%. Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Citrobacter and Flavobacterium were occasionally observed.     

Vessel occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field

Xylella fastidiosa is a Gram‐negative, xylem‐limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.     

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.     

Infiltration of Pseudomonas putida cells, strain 48, into xylem vessels of cut Rosa cv. 'Sonia'

Pseudomonas putida cells were unable to pass the inter-vessel pit membranes of the xylem system of cut roses ( Rosa hybrida cv. 'Sonia'). It was further shown that (1) the number of bacteria which infiltrated into the xylem vessels decreased with increased distance between the cutting point and sampling point; (2) the number of bacteria which infiltrated into the open xylem vessels increased with time and with increasing numbers of pseudomonas cells; (3) only a minor part of the pseudomonas cells homogeneously suspended in the vase solution was able to infiltrate into the xylem vessels of the cut roses up to a distance from the cutting point of > 1 cm; and (4) even low levels of infiltrated pseudomonas cells could be demonstrated by measurements of the water conductivity of stem segments. More research is needed to reveal which mechanisms (e.g. gumnosis) might have contributed, directly or indirectly, to the prevention of further infiltration of bacterial particles into the cut open vascular system of the Rosa cultivar.     

Mechanism of water stress-induced xylem embolism

We investigated the hypothesis that water stress-induced xylem embolism is caused by air aspirated into functional vessels from neighboring embolized ones (e.g. embolized by physical damage) via pores in intervessel pit membranes. The following experiments with sugar maple (Acer saccharum Marsh.) support the hypothesis. (a) Most vessels in dehydrating stem segments embolized at xylem pressures < −3 megapascals; at this point the pressure difference across intervessel pits between air-filled vessels at the segment's ends and internal water-filled vessels was >3 megapascals. This same pressure difference was found to be sufficient to force air across intervessel pits from air injection experiments of hydrated stem segments. This suggests air entry at pits is causing embolism in dehydrating stems. (b) Treatments that increased the permeability of intervessel pits to air injection also caused xylem to embolize at less negative xylem pressures. Permeability was increased either by perfusing stems with solutions of surface tension below that of water or by perfusion with a solution of oxalic acid and calcium. The mechanism of oxalic-calcium action on permeability is unknown, but may relate to the ability of oxalate to chelate calcium from the pectate fraction of the pit membrane. (c) Diameter of pores in pit membranes measured with the scanning electron microscope were within the range predicted by hypothesis (≤0.4 micrometer).     

Tyloses and ecophysiology of the early carboniferous progymnosperm tree Protopitys buchiana

Trunk woods of Early Carboniferous Protopitys buchiana show the earliest example of tylose formation and the first record for a progymnosperm. Protopitys tyloses are more densely located in inner trunk woods and near growth layer boundaries. We suggest, therefore, that an altered physiological state of living ray cells, during dormancy and/or following water stress, was necessary to make the woods vulnerable to tylose formation. Coupled with the distribution and proximity of abundant wood ray parenchyma to large xylem conducting cells, the positions of conduits filled with tyloses can be interpreted as ecophysiological responses of the plant to changes in local environment. In addition, some xylem conducting cells might have functioned as vessels. Fungal hyphae are present in some tracheary cells and in some areas with tyloses, but there is no evidence for wood trauma; we conclude, therefore, that these particular cases of tyloses are probably not induced by wound trauma. Protopitys buchiana wood thus shows structure/function similarities to modern woods with vessels, such as those of dicot angiosperms. This implies that ancient and modern plant ecophysiological responses correlate well with the physical parameters of their cellular construction.     

Lipids in xylem sap of woody plants across the angiosperm phylogeny

Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di- and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml⁻¹ across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.     

In vivo magnetic resonance imaging of xylem vessel contents in woody lianas

Previous reports suggest that in some plant species the refilling of embolized xylem vessels can occur while negative pressure exists in the xylem. The aim of this experiment was to use non‐destructive nuclear magnetic resonance imaging (MRI) to study the dynamics of xylem cavitation and embolism repair in‐vivo. Serial ¹H‐MRI was used to monitor the contents of xylem vessels in stems of two dicotyledonous (Actinidia deliciosa and Actinidia chinensis, kiwifruit) and one monocotyledonous (Ripogonum scandens, supplejack) species of woody liana. The configuration of the horizontal wide bore magnet and probe allowed the imaging of woody stems up to 20 mm in diameter. Tests using excised stems confirmed that the image resolution of 78 µm and digital image subtraction could be used to detect the emptying and refilling of individual vessels. Imaging was conducted on both intact plants and excised shoots connected to a water supply. In the case of Ripogonum the excised shoots were long enough to allow the distal end of the shoot, including all leaves, to be exposed to ambient conditions outside the building while the proximal end was inside the MRI magnet. In total, six stems were monitored for 240 h while the shoots were subjected to treatments that included light and dark periods, water stress followed by re‐watering, and the covering of all leaves to prevent transpiration. The sudden emptying of water‐filled vessels occurred frequently while xylem water potential was low (below ?0.5 MPa for Actinidia, ?1.0 MPa for Ripogonum), and less frequently after xylem water potential approached zero at the end of water‐stress treatments. No refilling of empty vessels was observed at any time in any of the species examined. It is concluded that embolism repair under negative pressure does not occur in the species examined here. Embolism repair may be more likely in species with narrower xylem vessels, but further experiments are required with other species before it can be concluded that repair during transpiration is a widespread phenomenon.     

Induction of air embolism in xylem conduits of pre-defined diameter.

A new method is presented that enables the induction of embolisms in a fraction of all xylem vessels, based on diameter, at one cut end of a stem segment. The method is based on the different capillary characteristic of xylem vessels of different cross-sectional size. To verify the method, air embolisms were induced in cut xylem vessels of chrysanthemum (Dendranthemaxgrandiflorum Tzvelev cv. Cassa) stem segments at different xylem tensions and compared with the distribution of gas-filled vessels as visualized by cryo-scanning electron microscopy (Cryo-SEM). At -6 kPa xylem pressure, air-entrance was only induced in large diameter vessels (>30 microm), while at -24 kPa embolisms were induced in almost all xylem vessels (>10 microm). Although the principle of the embolization method worked well, smaller diameter vessels were observed to be embolized than was expected according to the calculations. The role of cross-sectional shape and contact angle between xylem sap and vessel wall at the menisci are discussed. After correction for the observed (diameter independent) deviation from circularity of the cross-sectional vessel shape the contact angle was calculated to be approximately 55 degrees. Hydraulic resistance (Rh) measurements before and after embolization showed that the effect of embolizing only large diameter cut xylem vessels had only a small influence on overall Rh of a stem segment. Embolizing all cut xylem vessels at one cut end almost trebled overall Rh. The difference was discussed in the light of the networking capacity of the xylem system.     

PHYSIOLOGICAL STUDIES ON THE VERTICILLIUM WILT DISEASE OF TOMATO

The water loss per unit leaf area of tomato plants was decreased after inoculation with Verticillium albo-atrum. When diseased plants began to wilt water loss temporarily increased, but then rapidly decreased to become less than that of healthy plants grown under conditions of adequate or restricted water supply.
The transpiration of excised leaves from plants grown with a restricted water supply was reduced, but not so severely as that of comparable leaves from infected plants. Water loss from leaves on infected plants was reduced irrespective of any blocking of the petiolar xylem.
The rate of water loss from turgid leaf disks on mannitol solutions, and the rate of water uptake of leaf disks on water was similar for disks cut from wilting or turgid leaves of diseased plants or healthy plants grown with an adequate or restricted water supply.
Disease or poor water supply reduced leaf growth but had no effect on the rate of leaf initiation. Although the density of stomata was higher on leaves of diseased plants the stomatal area was less than on healthy plants.
The resistance to water flow in diseased stems was high and was correlated with vessel blockage. About half the blocked vessels contained hyphae. The severity and localization of symptoms in inoculated plants growing on susceptible or resistant rootstocks was directly related to the extent of invasion by the pathogen and to vessel blockage.
Experiments on the wilting activity of cell-free filtrates from cultures of the pathogen in vitro indicated that it produced a stable substance, not an enzyme, that caused wilting in cut shoots by blocking the end of the stem. It is suggested that an increasing internal water shortage causes major symptoms of disease.