Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5 end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.     

Nodule physiology of a supernodulating pea mutant

Physiological and biochemical parameters of the supernodulating pea (Pisum sativum L.) mutant nod₃ were compared to those of its wild-type parent cv. Rondo in a nil nitrate environment. Plants of cv. Rondo produced more biomass and accumulated more N than plants of nod₃. Accordingly, seed yield of the wild type was twice that of the supernodulating mutant. Although the nodule number of nod₃ was 10-fold that of cv. Rondo, the nodule mass of nod₃ was only twice that of cv. Rondo as individual nodules were smaller in nod₃ than in cv. Rondo. The maximum rate of acetylene reduction activity, determined in an open flow-through gas system, was higher in the wild type than in nod₃ when expressed on a nodule dry weight basis. However, when expressed on a whole plant basis, the nitrogenase activity (acetylene reduction) was similar in the two symbioses. The net carbon costs of nitrogenase activity was 25% lower in nod₃ than in cv. Rondo. An equal proportion of the net CO₂ efflux from the root system was for growth and maintenance of the tissue in the two symbioses. However, growth and maintenance respiration was higher in nod₃ than in cv. Rondo per gram dry weight of the nodulated root system. The nodules of nod₃ had a reduced soluble protein concentration as compared to those of the wild type. The specific activities of nodule glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14) and asparagine synthetase (EC 6.3.5.4) were lower in nod₃ than in cv. Rondo. The root bleeding sap of nod₃ contained lower amounts of glutamine and higher amounts of asparagine than that of cv. Rondo. The results suggest that the use of carbon directly related to the dinitrogen fixation and nitrogen assimilation may be less in nod₃ than in cv. Rondo, and that there may be differences between the two symbioses in the pathway for assimilation of fixed nitrogen.     

Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap

Roots of Pisum sativum L. and Zea mays L. were exposed to different moisture gradients established by placing both wet cheesecloth (hydrostimulant) and saturated aqueous solutions of various salts in a closed chamber. Atmospheric conditions with different relative humidity (RH) in a range between 98 and 86% RH were obtained at root level, 2 to 3mm from the water-saturated hydrostimulant. Roots of Silver Queen corn placed vertically with the tips down curved sideways toward the hydrostimulant in response to approximately 94% RH but did not respond positively to RH higher than approximately 95%. The positive hydrotropic response increased linearly as RH was lowered from 95 to 90%. A maximum response was observed at RH between 90 and 86%. However, RH required for the induction of hydrotropism as well as the responsiveness differed among plant species used; gravitropically sensitive roots appeared to require a somewhat greater moisture gradient for the induction of hydrotropism. Decapped roots of corn failed to curve hydrotropically, suggesting the root cap as a major site of hydrosensing.     

A chemically induced new pea (Pisum sativum) mutant SGECdt with increased tolerance to, and accumulation of, cadmium

BACKGROUND AND AIMS: To date, there are no crop mutants described in the literature that display both Cd accumulation and tolerance. In the present study a unique pea (Pisum sativum) mutant SGECd(t) with increased Cd tolerance and accumulation was isolated and characterized. METHODS: Ethylmethane sulfonate mutagenesis of the pea line SGE was used to obtain the mutant. Screening for Cd-tolerant seedlings in the M2 generation was performed using hydroponics in the presence of 6 microm CdCl2. Hybridological analysis was used to identify the inheritance of the mutant phenotype. Several physiological and biochemical characteristics of SGECd(t) were studied in hydroponic experiments in the presence of 3 microm CdCl2, and elemental analysis was conducted. KEY RESULTS: The mutant SGECd(t) was characterized as having a monogenic inheritance and a recessive phenotype. It showed increased Cd concentrations in roots and shoots but no obvious morphological defects, demonstrating its capability to cope well with increased Cd levels in its tissues. The enhanced Cd accumulation in the mutant was accompanied by maintenance of homeostasis of shoot Ca, Mg, Zn and Mn contents, and root Ca and Mg contents. Through the application of La(+3) and the exclusion of Ca from the nutrient solution, maintenance of nutrient homeostasis in Cd-stressed SGECd(t) was shown to contribute to the increased Cd tolerance. Control plants of the mutant (i.e. no Cd treatment) had elevated concentrations of glutathione (GSH) in the roots. Through measurements of chitinase and guaiacol-dependent peroxidase activities, as well as proline and non-protein thiol (NPT) levels, it was shown that there were lower levels of Cd stress both in roots and shoots of SGECd(t). Accumulation of phytochelatins [(PCcalculated) = (NPT)-(GSH)] could be excluded as a cause of the increased Cd tolerance in the mutant. CONCLUSIONS: The SGECd(t) mutant represents a novel and unique model to study adaptation of plants to toxic heavy metal concentrations.     

Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant ageotropum

In this study, ageotropum pea mutant was used to determine the threshold time for perception of an osmotic stimulation in the root cap and the time requirement for transduction and transmission of the hydrotropic signal from the root cap to the elongation region. The threshold time for the perception of an osmotic stimulation was compared to current estimates of threshold times for graviperception in roots. The time required for transduction and transmission in the hydrotropic response of ageotropum was compared to the time requirement in the gravity response of Alaska pea roots. We determined that threshold time for perception of an osmotic stimulation in the root cap is very rapid, occurring in less than 2 min following the application of sorbitol to the root cap. Furthermore, a single 5 min exposure of sorbitol to the root cap fully induced a hydrotropic response. We also found that transduction and transmission of an osmotic stimulus requires 90-120 min for movement from the root cap to more basal tissues involved in differential growth leading to root curvature. The very rapid threshold time for perception of root hydrotropism is similar to those times reported for root gravitropism. However, the time required for the transduction and transmission of an osmotic stimulation from the root cap is significantly longer than the time required in gravitropism. These results suggest that there must exist some differences between root hydrotropism and gravitropism in either the rate or mechanisms of transduction and transmission of the tropistic signal from the root cap.     

Roots of Pisum sativum L. exhibit hydrotropism in response to a water potential gradient in vermiculite

In the present study, root hydrotropism in an agravitropic mutant of Pisum sativum L. grown in vermiculite with a steep water potential gradient was examined. When wet and dry vermiculite were placed side by side, water diffused from the wet (-0.04 MPa) to the dry (-1.2 MPa) and a steep water potential gradient became apparent in the dry vermiculite close to the boundary between the two. The extent and location of the gradient remained stable between the fourth and sixth day after filling a box with vermiculite, and the steepest gradient (approx. 0.02 MPa mm-1) was found in the initially dry vermiculite between 60 and 80 mm from the boundary. When seedlings with 25-35 mm long roots were planted in the initially dry vermiculite near where the gradient had been established, each of the main roots elongated toward the wet vermiculite, i.e. toward the high water potential. Control roots elongated without curvature in both the wet and the dry vermiculite, in which no water potential gradient was detectable. These results show that pea roots respond to the water potential gradient around them and elongate towards the higher water potential. Therefore, positive hydrotropism occurs in vermiculite just as it does in air. Hydrotropism in soil may be significant when a steep water potential gradient is apparent, such as when drip irrigation is applied.     

Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant ageotropum.

In recent years, experiments have demonstrated that the gravity response of roots can be separated from the hydrotropic response by using the agravitropic pea mutant ageotropum. Though this mutant has been a useful tool for demonstrating the existence of the hydrotropic response of roots, little is known about how perception, transduction, transmission, and the growth response is accomplished. In this study, we have used the ageotropum mutant to investigate both the threshold time for perception of an osmotic stimulation and the minimum time required for signal transduction and transmission in roots following an osmotic stimulation at the root cap. In addition, we have compared the threshold times and signal transmission times of hydrotropism in the ageotropum roots to the gravity response of Alaska pea roots.     

Induction of hydrotropism in clinorotated seedling roots of Alaska pea,Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutantageotropum show positive hydrotropism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradlent. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropically and their weak hydrotropic response results from a counteracting effect of gravitropism.     

Abnormal root and nodule vasculature in R50 (sym16), a pea nodulation mutant which accumulates cytokinins

BACKGROUND AND AIMS: R50 (sym16) is a pea nodulation mutant with fewer and shorter lateral roots (LR), fewer nodules and high levels of cytokinins (CK). Because a link exists between CK imbalance and abnormal vasculature, the vasculature of the primary root (PR) and LR of R50 was studied and it was compared with that of the wild-type 'Sparkle'. Also nodule vasculature was investigated to correlate R50 low nodulation phenotype with CK accumulation. METHODS: PR and first-order LR were hand-sectioned transversely in different locations and at different ages. Vascular poles were counted and root and stele diameters measured. To evaluate LR primordia number, roots were cleared. Nodules obtained from inoculated plants were either fixed and sectioned or cleared; numbers of vascular strands and of tracheary elements in the strands were counted. KEY RESULTS: 'Sparkle' PR is triarch, whereas that of R50 can be triarch, tetrarch or pentarch. Furthermore, as the R50 roots developed, supernumerary vascular strands appeared but, as they aged, the new growth of more roots displayed the triarch pattern. LR vasculature differed from that of PR: whereas 'Sparkle' LR had three or four poles, those of R50 had two or three. No differences in PR or PR stele diameters existed between the two lines. Whereas 'Sparkle' nodules had two vascular strands, most R50 nodules possessed three; however, because R50 nodules were variable in size, their vasculature was highly diverse in terms of strand length. A strong correlation was found between nodule length and number of tracheary elements in strands. CONCLUSIONS: R50 displays an additional number of vascular poles in its PR, a smaller number of vascular poles in its first-order LR and an altered vasculature in its nodules. It appears that these three characteristics are linked to the high levels of CKs that the mutant accumulates over its development.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.     

The nature of protein differentiation in the growing pea root

Abstract Sections of the growing root of pea (Pisum sativum) have been microdissected into stele, cortex and epidermis. Using labelled amino acids, two dimensional separations employing non-equilibrium pH gel electrophoresis (NEPHGE) and isoelectric focusing (IEF), and silver staining, the complexity of protein differences between the cortex and the stele has been assessed. Analyses commenced as cells in these two tissues appear in the meristem (0.7—1 mm from the tip) and continued up to 30 mm from the tip as they subsequently mature. From the earliest stages at which the cortex and stele can be distinguished and dissected apart the protein patterns differ substantially. However these tissue differences, involving over one third of the detected protein species, are almost all quantitative. Very few qualitative (i.e. tissue specific) proteins were detected. Many proteins also show quantitative stage-specific variation, detected using successively older root segments. In vitro translation studies involving isolated mRNA showed only a very limited stage-specific variation in translated proteins. This supports the notion that translational controls may contribute significantly to the development of these two tissue types.     

Indole-3-acetaldehyde oxidase of pea seedlings

Indole-3-acetaldehyde oxidase was partially purified from the epicotyl of Pisum satiyum seedlings by column chromatography using CM-Sephadex and Sephadex G-150. The enzyme was only active in the presence of molecular oxygen. The activity was maximal at pH 8.0, and the Km value for indole-3-acetaldehyde was 1.4 × 10⁻³ M . The enzyme was inhibited strongly by p -hydroxymercuribenzoate, cyanide and hydroxylamine, suggesting that it contains sulfhydryl group(s) and a metal component such as iron.     

Maximum axial root growth pressure in pea seedlings: effects of measurement techniques and cultivars

Values of the maximum axial growth pressure (σ_max) of seedling pea (Pisum sativum L.) roots reported in the literature, obtained using different apparatuses and cultivars, range from 0.3 MPa to 1.3 MPa. To investigate possible reasons for this large range, we studied the effect of apparatus and cultivar on measurements of σ_max in peas. We describe four types of apparatus which can be used to measure axial root growth force and hence σ_max, and used them to measure σ_max in seedling pea roots using cultivar Meteor. Two of these apparatuses were also used to compare σ_max for three pea cultivars (Helka, Meteor and Greenfeast). Both cultivar and apparatus significantly affected σ_max , but there were greater differences between apparatuses than between the three cultivars. Estimating root cross-sectional area from the diameter of cross-sections, rather than from in situ measurements (i.e. measurements made with the root still in place in the apparatus) may explain these differences. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple protocol to purify fresh nuclei from milligram amounts of meristematic pea root tissue for biochemical and flow cytometry applications

A simple protocol to purify fresh nuclei from very small amounts (mg) of Pisum sativum (cv. Lincoln) root tissue is presented. The protocol is reliable and has been repeated many times; it can be performed within a few hours and needs only a simple modification of common glassware for tissue homogenization. Similar yields of purified nuclei are obtained both with meristematic and adult root tissues using a detergent-free glyeerol buffer. Purified nuclei can be stored at –20°C for several weeks without any appreciable loss of integrity. The high yield of purified nuclei enables us to use our preparations for biochemical and cytometric investigations.     

Water potential, turgor and cell wall properties in elongating tissues of the hydrotropically bending roots of pea (Pisum sativum L.)

The hydrotropic bending of roots of an ageotropic pea mutant, ageotropum, was studied in humid air in a chamber with a steady humidity gradient. We examined the effects of atmospheric humidity around the root on the water status of root tissues, as well as the wall growth and the hydraulic properties of the elongating tissues. Atmospheric humidity at the surface of the root was clearly lower on the side orientated towards the air with lower humidity than on the side orientated towards the air with higher humidity. However, there were no differences in water potential and osmotic potential between the tissues that faced air with higher and lower humidities in the elongating and mature regions. Plastic extensibility was higher in the tissues that faced the air with lower humidity than in the tissues that faced the air with higher humidity. No differences in turgor pressure and yield threshold were observed between the tissues that faced air with higher and lower humidities. Therefore, the extensibility of the cell wall appeared to be responsible for the different growth rates of tissues in root hydrotropism. A further probable cause of the hydrotropical bending of roots is changes in the hydraulic conductance in the elongating tissues. Since the hydrotropic bending of roots occurred only when a root tip was exposed to a humidity gradient, hydrotropism might occur after perception of a difference in humidity by the root tip, with accompanying changes in cell wall extensibility and hydraulic conductance.     

Lipid peroxidation in and photo-damage to a light-sensitive chlorescence pea mutant

Leaves of 10- to 12-day-old chlorescence lethal Pisum sativum L. mutant are similar to control plants with respect to the content of chlorophylls, carotenoids, fatty acids and α-tocopherol. Subsequent development of the mutant under high irradiation resulted in th destruction of the photosynthetic pigments, polyunsaturated fatty acids, α-tocopherol, and also in the accumulation of liposoluble fluorescent products. No increase in the level of malondialdehyde was observed. In chloroplasts isolated from mutant plants the contents of chlorophyll a and β-carotene were decreased to a greater extent than the more oxidized pigments (xanthophylls and chlorophyll b ). The data obtained are discussed with special reference to the role of lipid peroxidation in the injury of plant cells under the action of visible light and to the antioxidative mechanisms stabilizing photosynthetic membranes.     

Neutral growth inhibitors in light-grown dwarf pea shoots –separation, identification and growth regulation

To correlate endogenous growth inhibitors of dwarf pea to its dwarfism a thorough search of growth inhibitors was made in the neutral fraction of an acetone extract from the shoots of light-grown seedlings of Pisum sativum L. cvs Progress No 9 and Alaska. From both cultivars six inhibitors were separated and named A-1, A-2, A-3, B-1, B-2 and B-3 based on their order of elution from the silica gel column. Their contents as determined by cress root bioassay were: in cv. Progress 0.40, 16.5, 6.36, 1.02, 0.11 and 0.10, and in cv. Alaska 0.33, 2.35, 3.51, 0.95, 0.10 and 0.09 cress units (g fresh weight)⁻¹. Their contributions to growth regulation of the relevant pea plants were estimated as the products of the above-stated contents times the ratios of the specific activities of each standard sample in the cress roots and the relevant pea cultivars, and they were; in cv. Progress A-2, 5.44 and A-3, 2.10, and in cv. Alaska A-2, 0.68 and A-3, 0.88, those of A-2 and A-3 constituting more than 90% of the total contribution of the six inhibitors in either cultivar. The great differences in the content and contribution to growth regulation of A-2 and A-3 between the two cultivars suggest that the higher contents of and greater responsiveness to the two inhibitors in cv. Progress may be causes of dwarfism of this cultivar. A-l and A-3 were spectroscopically identified with pisatin and a mixture of cis, trans– , and trans, trans xanthoxins.     

Regeneration systems from immature embryos of Bulgarian pea genotypes

Ten genotypes from Pisum sativum and Pisum arvense were screened for their regeneration abilities. Most of them were created through experimental mutagenesis from Bulgarian varieties and have various valuable agronomic traits. Embryonic axes from immature embryos were plated on modified Murashige and Skoog medium, containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d), α-naphthaleneacetic acid (NAA) and benzyladenine (BA). Two schemes for direct and indirect organogenesis were established. Callus and shoot formation were induced on media containing 0.2 mM 2,4-d or 5 mM BA, respectively. Embryonic axes formed buds directly when plated on medium with 10 mM BA and 1 mM NAA. Organogenesis and adventitious bud formation were maintained on medium supplemented with BA and NAA. Rhizogenesis was induced on Gamborgs' B5 medium. All screened genotypes were able to regenerate plants with a high efficiency (50–100%) although some differences in their organogenetic response were observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.