The relationship between wound-induced proteinase inhibitors and hydraulic signals in tomato seedlings

Localized wounding is known to induce systemic proteinase inhibitors (PI) in seedlings of tomato (Lycopersicon esculentum L.). Inhibitors of elastase (EC 3.4.21.36) were shown here to be among those systemically induced by wounding, and a simple rapid assay for PI based on elastase was developed. Using this assay, the nature of the systemic signalling system (‘PIIF’) was investigated. Hydraulic signals were shown to be induced in tomato by localized wounds. These signals travelled throughout the plant well within the lag time before appearance of systemic wound-induced PI. A number of correlations were drawn between the occurrence of the hydraulic signals and induction of systemic PI, suggesting that hydraulic signals might be the PIIF, or a component of it. It was shown that systemic hydraulic signals could be triggered, without significant wounding, by excision of a single leaflet through the submerged petiole. These hydraulic signals were similar in both kinetics and magnitude to those induced by localized wounding. However, they did not induce systemic PI. In addition, it was shown that systemic events almost as rapid as wound-induced hydraulic signals could be induced without wounding, under certain environmental conditions. This indicates that rapid hydraulic signals do not provide a specific signal of wounding. These findings demonstrate that hydraulic signals per se are not the PIIF.     

Kinetics of wound-induced hydraulic signals and variation potentials in wheat seedlings

Displacement transducers were used to demonstrate that localised wounding causes a rapid and systemic increase in leaf thickness in seedlings of wheat (Triticum durum Desf. cv. Iva). These increases are interpreted as reflecting wound-induced hydraulic signals. The duration of the wound-induced increase was found to be about 1 h or more, and it was shown that repeated wounds could induce repeated responses. The increase occurred even when plants had no access to an external water supply. Change in leaf thickness was shown closely to reflect change in leaf water potential. The velocity and kinetics of the wound-induced hydraulic signal were measured using multiple transducers ranged along a single leaf. The front of the signal was shown to travel through the plant at rates of at least 10 cm · s^–1. Development of the increase in leaf thickness was found to be relatively faster furthest from the wound. Onset of the change in leaf thickness in leaves remote from the wound was shown to precede onset of changes in surface electrical potential (variation potential) which are also induced by wounding. In contrast to reports from other species, variation potentials in wheat were here shown to spread extremely rapidly, at rates similar to that of the hydraulic signal. These data support the view that wound-induced hydraulic signals are the trigger for variation potentials in wheat.Symbol: _w water potential Grateful thanks are due to Paul Springer of the HRI (Wellesbourne) mechanical workshop for building equipment, and to H.G. Jones for helpful discussion.     

Pressure-volume curves and drought resistance in two wheat genotypes

The water relations of two durum wheat cultivars ( Triticum durum Desf.) were studied throughout the growing season. Irrigated and unirrigated plants were compared from booting to milk stage; a period where water stress occurred naturally in the field. Modulus of elasticity (ε), turgid weight/dry weight ratio (TW/DW), relative water content at zero turgor (RWC_o) and osmotic potential at full turgor (ε) declined throughout the season while average turgor (ψ_p) increased. Water stress induced a further decrease in ψ_π¹⁰⁰ and the TW/DW ratio. The elastic modulus varied greatly. During the first stages of growth, cv. Appulo (the more resistant cultivar) showed lower ε values than cv. Valforte. At the milk stage, ε was lower for the unirrigated than the irrigated plants. Correlation coefficients between the TW/DW ratio and the osmotic potential were significant for both cultivars. In cv. Valforte, TW/DW was also correlated with the average turgor and the bulk modulus of elasticity. Structural changes that affect the TW/DW ratio seem to be important factors influencing water relations and drought tolerance in durum wheat.     

Leaf hygrometer v. pressure chamber: a comparison of pressure–volume curve data obtained on single leaves by alternating measurements

Abstract. Data for the construction of pressure-volume curves were obtained by measuring water potentials of detached leaves repeatedly and alternately, with a pressure chamber and a leaf hygrometer. Good agreement between the parameters of the two resulting curves was observed. Regression lines on values after the loss of turgor were always more negative for the thermocouple data, with a maximum difference for the osmotic potential at full saturation of 0.25 MPa in Triticum and a minimum of 0.01 MPa in Populus. Neither the slopes of the regression lines nor the intercepts with the axes were statistically different. We see no reason for using one of these two unrelated methods as a standard against which the other is calibrated. Implications for the theory of pressure-volume curves are discussed.     

Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics

Sunflower plants ( Helianthus annuus L.) were given an electrical stimulus to the stem or a heat (flame)‐wound to a single leaf or a cotyledon. The resulting electrical activity was monitored with extracellular electrodes. An electrical stimulus applied to the stem frequently evoked an action potential (AP), but never a variation potential (VP). In contrast, a heat‐wound applied to a leaf virtually always elicited a VP, which was often accompanied by one or more superimposed spikes (putative APs). The kinetic parameters of the AP and the VP were investigated. The AP appears to propagate without decrement in velocity or magnitude, whereas the VP parameters decrease significantly with distance. The heat stimulus triggered rapid alterations in stem elongation/contraction, which preceded changes in electrical potential, indicating the transmission of a hydraulic signal. Light‐off and light‐on stimuli evoked negative‐ and positive‐going changes in extracellular electrical potential, respectively, corresponding to de‐ and hyper‐polarization of the plasma membrane. Membrane depolarization (extracellularly manifested as a VP) evoked by both the light‐off and heat‐wounding stimuli was able to trigger one or more APs. We interpret these results to suggest that APs are "genuine" electrical signals involving voltage‐gated ion channels or pumps, which can be evoked directly by electrical stimulation or indirectly by changes in membrane potential occurring during the VP or after the light‐off stimulus. In contrast, VPs appear to be a local (non‐transmissible) electrical consequence of the passage of a rapidly transmitted hydraulic signal in the xylem, presumably acting on mechanosensitive ion channels or pumps in adjacent living cells.     

Uptake of zinc by rye,bread wheat and durum wheat cultivars differing in zinc efficiency

Effect of zinc (Zn) nutritional status on uptake of inorganic ⁶⁵Zn was studied in rye (Secale cereale, cv. Aslim), three bread wheat (Triticum aestivum, cvs. Dagdas, Bezostaja, BDME-10) and durum wheat (Triticum durum, cv. Kunduru-1149) cultivars grown for 13 days in nutrient solution under controlled environmental conditions. The cultivars were selected based on their response to Zn deficiency and to Zn fertilization in calcareous soils under field conditions. When grown in Zn-deficient calcareous soil in the field, the rye cultivar had the highest, and the durum wheat the lowest Zn efficiency. Among the bread wheats, BDME-10 showed higher susceptibility to Zn deficiency and Bezostaja and Dagdas were less affected by Zn deficiency. Similarly to field conditions, in nutrient solution visual Zn deficiency symptoms (i.e. necrotic lesions on leaf blade) appeared to be more severe in Kunduru-1149 and BDME-10 and less severe in rye cultivar Aslim. Under Zn deficiency, shoot concentrations of Zn were similar between all cultivars. Cultivars with adequate Zn supply did not differ in uptake and root-to-shoot translocation rate of ⁶⁵Zn, but under Zn deficiency there were distinct differences; rye showed the highest rate of Zn uptake and the durum wheat the lowest. In the case of bread wheat cultivars, ⁶⁵Zn uptake rate was about the same and not related to their differential Zn efficiency. Under Zn deficiency, rye had the highest rate of root-to-shoot translocation of ⁶⁵Zn, while all bread and durum wheat cultivars were similar in their capacity to translocate ⁶⁵Zn from roots to shoots. When Zn²⁺ activity in uptake solution ranged between 117 p M and 34550 pM, Zn-efficient and Zn-inefficient bread wheat genotypes were again similar in uptake and root-to-shoot translocation rate of ⁶⁵Zn. The results indicate that high Zn efficiency of rye can be attributed to its greater Zn uptake capacity from soils. The inability of the durum wheat cultivar Kunduru-1149 to have a high Zn uptake capacity seems to be an important reason for its Zn inefficiency. Differential Zn efficiency between the bread wheat cultivars used in this study is not related to their capacity to take up inorganic Zn. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isoprenylation modification is required for HIPP1-mediated powdery mildew resistance in wheat

Powdery mildew (Pm), caused by Blumeria graminis f.sp. tritici (Bgt), is one of the most important wheat diseases. Heavy-metal-associated isoprenylated plant protein (HIPP1) has been proved playing important roles in response to biotic and a biotic stress. In present study, we proved HIPP1-V from Haynalidia villosa is a positive regulator in Pm resistance. HIPP1-V was rapidly induced by Bgt. Transiently or stably heterologous overexpressing HIPP1-V in wheat suppressed the haustorium formation and enhanced Pm resistance. HIPP1-V isoprenylation was critical for plasma membrane (PM) localization, interaction with E3-ligase CMPG1-V and function in Pm resistance. Bgt infection recruited the isoprenylated HIPP1-V and CMPG1s on PM; blocking the HIPP1 isoprenylation reduced such recruitment and compromised the resistance of OE-CMPG1-V and OE-HIPP1-V. Overexpressing HIPP1-VC^148G could not enhance Pm resistance. These indicated the Pm resistance was dependent on HIPP1-V's isoprenylation. DGEs related to the ROS and SA pathways were remarkably enriched in OE-HIPP1-V, revealing their involvement in Pm resistance. Our results provide evidence on the important role of protein isoprenylation in plant defense.     

Comparison of electric and growth responses to excision in cucumber and pea seedlings. I. Short-distance effects are a result of wounding

The local electric response to stem excision in both pea epicotyls and cucumber hypocotyls is a depolarization of the cells in the wound area. If we define wound area as the region of local depolarization, we find that it extends for approximately 10 mm from the cut or wound site in pea epicotyls, whereas it can reach up to 40 mm in cucumber hypocotyls. The wound-induced depolarization in pea cells is transient, reaching its maximal amplitude within 1–2 min, whereas in cucumber cells this depolarization is more sustained. A third difference between wound responses in pea and cucumber is the intermittent appearance of spikes, i.e. very short, rapidly reverted depolarizations which frequently accompany the basic depolarization in cucumber but not in pea cells. These spikes can propagate in both directions along the hypocotyl axis. The cause of the different responses of pea and cucumber cells is unknown. A possible explanation might be found in different degrees of electrical cell coupling in the two species. This possibility was investigated in cucumber hypocotyls by measuring the cell input resistance (R_in) of epidermal cells at various axial distances from the cut. Shorter distances increase the likelihood of shunting the cell membrane resistance through the shortened symplastic path to the cut surface. With a series of cuts made at decreasing distances from the measured site, cell depolarization increased without comparable changes in R_in. Two conclusions were drawn. Firstly, wound-induced depolarizations are not brought about by shunting of the cell resistance in the wound area. Secondly, the depolarization is probably not carried by ion channels but may be caused by an inhibition of proton pump activity. Parallel to its depolarizing effect on the membrane potential, excision led to a severe and sustained decline in the cucumber hypocotyl growth rate only when carried out sufficiently close to the growing region (45 mm from the hook). Similar excision in pea epicotyls failed to change the growth rate. Both electrical and growth data support the concept that the high and sustained responsiveness of cucumber seedlings to wounding is caused by a particular sensitivity of their proton pump mechanism.     

春小地片质膜氧化还原系统及其对缓慢干旱胁迫的响应

研究了抗旱性不同的2个品种小麦（Triticum aestivum L.)叶片质膜氧化还原系统的部分性南及其在田间缓慢干时时下氧化还原活力的变化。结果显示,2个品种小麦叶片质膜氧化还原活性的最适pH为8．0,最适温度在40℃左右,Mg^2 对其活性有刺激作用,Ca^2 对其活性没有影响。但这2个品种叶片的质膜氧化还原系统对K^ 和Na^ 的响应不尽相同：在品种定西24中,K^ 刺激作用不太明显,Na^ 有一定的抑制作用;而在品种8139中,这两种离子都有明显的刺激作用。干旱降低了小麦叶片的水势和水分含量,影响了小麦的生长发育;在缓慢干旱下,小麦叶片质膜氧化还原活力在生长发育的前期上升;在后期,其活性不变或下降,这与前人在实验室内以植物幼苗进行短期而剧烈的模拟干旱下所观察的结果不同。这种差异的原因除了与植物材料不同有关外,主要与胁迫方式及植物的发育阶段有关。     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

The interaction between salinity and boron toxicity affects the subcellular distribution of ions and proteins in wheat leaves

Salinity aggravates B toxicity symptoms in several plant species. In the present study the interactive effects of B toxicity and salinity stresses on the subcellular distribution of boron, cations and proteins in basal and apical leaf sections of wheat were investigated. High B supply increased total B concentrations in all leaf parts, but values remained below 25 mg B kg^?1 dry weight (DW) in basal sections, whereas they reached more than 600 mg B kg^?1 DW in leaf tips. In basal leaf sections intercellular soluble B concentrations closely reflected the external supply, whereas intracellular soluble B concentrations remained lower by a factor of two, indicating some retention of excess B in the apoplast. Combined salinity and B toxicity stresses significantly increased soluble B concentrations in inter‐ and intracellular compartments of basal leaf sections in comparison with either stress alone, probably related to salinity‐induced changes in water status. The combined stresses also induced quantitative and qualitative changes in inter‐, but not intracellular protein composition. Most obvious was the induction of a 25 kDa protein and an increase in amount of a 33 kDa protein. It is suggested that these changes might be due to structural modifications of the cell wall. The concentration of soluble boron in cells is proposed to be an indicator of boron toxicity.     

Effect of light on abscisic acid‐induced proline accumulation in leaves: comparison between barley and wheat

Light enhanced the abscisic acid‐induced accumulation of proline in barley ( Hordeum vulgare L. cv. Georgie) and wheat ( Triticum durum L. cv. Valnova). In wheat ABA is ineffective in the dark. In both barley and wheat, the accumulation of proline in the light showed the same characteristics as those of the process that occurs in barley in the dark, namely a synergistic interaction between the hormone and K(Na)Cl, an enhancing effect of Cl⁻ anion in excess over K⁺ cation in the incubation medium, and an inhibiting effect of D ‐mannose and monensine. In wheat, furthermore, light is needed during treatment with ABA if proline is to accumulate. Light was effective in both wheat and barley during the second or accumulation phase of the hormonal process, whereas the events occurring in the first (or lag) phase did not require light. The results suggest that in wheat light induces a putative factor(s) involved in the proline accumulation pathway that is lost in the dark, whereas in barley it is present in the dark.     

Changes in water relations and endogenous abscisic acid content of wheat and barley grains and embryos during development

Abstract. Immature cereal embryo development can be controlled by in vitro culture on media containing ABA, or by media of low osmotic potential. To assess the possible in vivo roles of these factors, endogenous ABA levels and water relations of embryos and grains of wheat ( Triticum aestivum L.) and barley ( Hordeum vulgare L.) were determined during development. ABA concentrations remained consistent with those required to inhibit precocious germination in vitro of early stage embryos but not of more mature embryos. With increasing maturity, a difference in water potential developed between grain and embryo, suggestive of an in vivo role for water status in controlling the development of the embryo.     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

Water-relation parameters of giant and normal cells of Capsicum annuum pericarp

Abstract. Measurements of the water-relation parameters of the giant subepidermal cells (volume, V = 0.119 to 1.658 mm³; = 0.53±0.35 mm³, SD, n = 23) and the smaller mesocarp parenchyma cells ( V = 0.10 to 0.79×10⁻³ mm³; = 0.36±0.27×10⁻³ mm³, SD, n = 6) of the inner pericarp surface of Capsicum annuum L. were made using the Jülich pressure probe. The volumetric elastic modulus ɛ for the large cells was between 1.5 and 27 MPa for a pressure range of 0.09 to 0.41 MPa. For the small cells ɛ was 0.1 to 0.6 MPa for a pressure range of 0.22 to 0.39 MPa. The turgor pressure P , the half-time of water exchange T _1/2, and the hydraulic conductivity L _p were as follows, with SD and number of replicates: large cells, P = 0.27±0.06 MPa (23), T _1/2=2.7±2.2 s (46), L _p=5.8±3.7 pm s⁻¹ Pa⁻ (46); small cells, P = 0.33±0.07 MPa (6), T _1/2= 33±10s (12), L _p=0.21±0.07 pm s⁻¹ Pa⁻¹ (12). The determination of these basic water-relation parameters is considered as a prerequisite for future ecotoxicological and phytopathological studies. The differences between the large and the small cells are discussed in relation to a desirable biophysical definition of succulence. Further, for the large cells a pressure and volume dependence of ɛ was demonstrated.     

Effect of nickel on ROS content and antioxidative enzyme activities in wheat leaves

Influence of 100 μM Ni on growth, Ni accumulation,, H₂O₂ and lipid peroxides contents as well as the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and glutathione peroxidase (GSH-Px) were studied in the leaves of wheat plants on the 3rd, 6th and 9th days after treatment. Exposure of the plants to Ni for only 3 days led to almost 200-fold increase in this metal concentration in the leaf tissue but later the rate of Ni accumulation was much slower. Length and fresh weight of the leaves were substantially reduced, up to 25% and 39%, respectively at the end of experiment. Visible symptoms of Ni toxicity: chlorosis and necrosis were observed following the 3rd day. Treatment with Ni resulted in the increase in and H₂O₂ contents in the leaves. Both showed their highest values, approximately 250% of those of the control, on the 3rd day and then their levels decreased but still markedly exceeded the control values. SOD and CAT activities decreased significantly in response to Ni treatment, however a several-fold increase in APX and POD activities was found. No significant changes in lipid peroxides content were observed in the leaves after Ni application. The activity of GSH-Px showed a 29% induction on the 3rd day. Our results indicated that despite prolonged increases in and H₂O₂ levels, oxidative damage, measured as the level of lipid peroxidation, did not occur in the leaves of Ni-treated wheat.     

Local and systemic changes in gene expression induced in tomato plants by wounding and by elicitor treatment

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants have been wounded to induce the accumulation of proteinase-inhibitor proteins (PI proteins) at the local site of injury and systemically in unwounded tissues. To determine the range of genes affected in the wound-response, polysomal mRNA has been isolated from the damaged leaves and from systemically responding leaves over a time-course of 2, 4, 10 and 24 h after wounding. Changes in the pattern of ³⁵S-translation products indicate that the events that occur at the local wound-site are different from those that occur systemically, both with respect to the number of genes that are regulated and the timing of their regulation. In order to compare the effects of wounding and an endogenous systemic signal generated at the wound-site with those of elicitor (proteinase-inhibitor-inducing factor, PIIF) treatment of excised plants, polysomal mRNA has also been isolated from leaves of plants over a time-course of 2, 4, 10 and 24 h after PIIF-treatment. Changes in the pattern of ³⁵S-translation products indicates that the events induced by PIIF resemble those induced by mechanical injury, rather than those induced by the endogenous systemic signal.Abbreviations IFF isoelectric focussing - PI proteins proteinase inhibitor proteins - PIIF proteinase-inhibitor-inducing factor - ssRubisco small subunit of ribulose-1,5-bisphosphate carboxylase     

Gradients and dynamics of inner bark and needle osmotic potentials in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst)

Preconditions of phloem transport in conifers are relatively unknown. We studied the variation of needle and inner bark axial osmotic gradients and xylem water potential in Scots pine and Norway spruce by measuring needle and inner bark osmolality in saplings and mature trees over several periods within a growing season. The needle and inner bark osmolality was strongly related to xylem water potential in all studied trees. Sugar concentrations were measured in Scots pine, and they had similar dynamics to inner bark osmolality. The sucrose quantity remained fairly constant over time and position, whereas the other sugars exhibited a larger change with time and position. A small osmotic gradient existed from branch to stem base under pre‐dawn conditions, and the osmotic gradient between upper stem and stem base was close to zero. The turgor in branches was significantly driven by xylem water potential, and the turgor loss point in branches was relatively close to daily minimum needle water potentials typically reported for Scots pine. Our results imply that xylem water potential considerably impacts the turgor pressure gradient driving phloem transport and that gravitation has a relatively large role in phloem transport in the stems of mature Scots pine trees.