Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips

The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development.     

Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies

Relations between shoot to root dry weight ratio (S : R), total plant dry weight (DW), shoot and plant N concentration and leaf soluble protein concentration were examined for pea ( Pisum sativum L.), common bean ( Phaseolus vulgaris L.) and wheat ( Triticum aestivum L.) under different nutrient deficiencies. A regression model incorporating leaf soluble protein concentration and plant DW could explain greater than 80% of the variation in S : R within and between treatments for pea supplied different concentrations of NO₃^– or NH₄⁺ in solid substrate; pea and bean supplied different concentrations of N, P, K and Mg in liquid culture; and wheat supplied different concentrations of N, P, K, Mg, Ca and S in liquid culture. Addition of shoot or plant N concentration to the model explained little more of the variation in S : R. It is concluded that results are consistent with the proposal that macronutrient effects on S : R are primarily mediated through their effects on protein synthesis and growth.     

Comparative Analysis of Triticum aestivum L. (Poaceae,Poales) and Pisum sativum L. (Fabaceae,Fabales) Resistance to Heavy Metals

Biology Bulletin - Abstract—Heavy metals are widespread soil pollutants frequently identified in agricultural lands. In many cases, their pollution levels significantly exceed the maximum...     

Design, expression, and crystallization of recombinant lectin from the garden pea (Pisum sativum)

The propeptide form of the lectin from the garden pea (Pisum sativum agglutinin) has been expressed in Escherichia coli by attaching its cDNA to an inducible promoter. By a number of criteria, including the ability to form dimers, hemagglutination titer, Western blot, and enzyme-linked immunosorbent assay, the resulting propeptide molecule is virtually indistinguishable from the mature proteolytically processed lectin isolated from peas. Preliminary crystallization experiments using the recombinant propeptide lectin yield crystals in space group P2(1)2(1)2(1) with a = 64.8 A, b = 73.8 A, and c = 109.0 A (1 A = 0.1 nm) that diffract to 2.8-A resolution. This unit cell size is quite similar to the unit cell determined for native pea lectin, suggesting that the overall structure of the recombinant prolectin is virtually identical.     

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.     

cDNA cloning, primary structure and gene expression for H-protein, a component of the glycine-cleavage system (glycine decarboxylase) of pea (Pisum sativum) leaf mitochondria.

We have isolated and characterized cDNA clones encoding the H-protein of the glycine-cleavage system of pea (Pisum sativum) leaf mitochondria. The deduced primary structure revealed that the 131-amino-acid polypeptide is cytoplasmically synthesized with a 34-amino-acid mitochondrial targeting peptide. The lipoate-binding site was assigned to be lysine-63, as deduced from a sequence comparison with several lipoate-bearing proteins. The expression of the gene encoding H-protein was shown to occur specifically in the leaf tissue, with light exerting an additional effect by increasing the mRNA levels severalfold. Two polyadenylation sites were found in the mRNA, and a single-copy gene encoding the H-protein was detected in pea genome.     

Gravity-controlled asymmetrical transport of auxin regulates a gravitropic response in the early growth stage of etiolated pea (Pisum sativum) epicotyls: studies using simulated microgravity conditions on a three-dimensional clinostat and using an agravitropic mutant, ageotropum

Increased expression of the auxin-inducible gene PsIAA4/5 was observed in the elongated side of epicotyls in early growth stages of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown in a horizontal or an inclined position under 1 g conditions. Under simulated microgravity conditions on a 3D clinostat, accumulation of PsIAA4/5 mRNA was found throughout epicotyls showing automorphosis. Polar auxin transport in the proximal side of epicotyls changed when the seedlings were grown in a horizontal or an inclined position under 1 g conditions, but that under clinorotation did not, regardless of the direction of seed setting. Accumulation of PsPIN1 and PsPIN2 mRNAs in epicotyls was affected by gravistimulation, but not by clinorotation. Under 1 g conditions, auxin-transport inhibitors made epicotyls of seedlings grown in a horizontal or inclined position grow toward the proximal direction to cotyledons. These inhibitors led to epicotyl bending toward the cotyledons in seedlings grown in an inclined position under clinorotation. Polar auxin transport, as well as growth direction, of epicotyls of the agravitropic mutant ageotropum did not respond to various gravistimulation. These results suggest that alteration of polar auxin transport in the proximal side of epicotyls regulates the graviresponse of pea epicotyls.     

Genetic variation in pea (Pisum) dehydrins: sequence elements responsible for length differences between dehydrin alleles and between dehydrin loci in Pisum sativum L.

 The electrophoretic patterns of dehydrins extracted from mature seeds of a range of pea (Pisum) species revealed extensive variation in dehydrin polypeptide mobility. Variation was also observed among lines of P. sativum. Crosses between lines with different dehydrin electrophoretic patterns produced F₁ seeds with additive patterns, and segregation in the F₂ generation was consistent with a 1 : 2 : 1 ratio, indicating allelic variation at each of two dehydrin loci (Dhn2, Dhn3). Genetic linkage was observed between Dhn2 and Dhn3, and the segregation ratios indicated preferential transmission of one allele at the Dhn3 locus. Dehydrin cDNA clones were characterised that encoded the allelic variants at Dhn2 and Dhn3. Their deduced amino-acid sequences were very similar to each other as well as to the product of the Dhn1 locus reported previously. Comparisons were made between the sequences of allelic variants at a single locus, and between the products of different loci. Differences in the electrophoretic mobilities between allelic variants at Dhn2 and Dhn3 were associated with differences in polypeptide length resulting principally from tandem duplications of 21 (Dhn2) or 24 (Dhn3) amino-acid residues. These duplications accounted for much of the difference in length between dehydrins encoded by the different loci. The conserved core of one of the duplicated regions varied in copy number, and small insertions/deletions of amino acids near this core also contributed to length variation both between allelic forms and between loci. Dehydrins possess characteristic highly conserved amino-acid sequence motifs, yet vary considerably in length. Mechanisms involving sequence duplication appear to be responsible for generating the length differences observed between allelic variants as well as between the products of different loci. Received: 12 June 1997 / Accepted: 29 October 1997     

Correlation between the inhibition of photosynthesis and the decrease in area of detached leaf discs or volume/absorbance of protoplasts under osmotic stress in pea (Pisum sativum)

Exposure to osmotic stress reduces leaf area and protoplast volume while decreasing photosynthesis. But the measurement of protoplast volume is tedious, while rapid determinations of leaf area in the field are difficult. We evaluated the quantitative relationship between the extent of decrease in area of detached leaf discs or the volume of protoplast of pea ( Pisum sativum ) and reduction in their photosynthetic capacity under osmotic stress. Osmotic stress was induced by increasing sorbitol concentration in the surrounding medium of the leaf discs from zero to 1.0 M (-3.1 MPa), and in case of protoplasts from 0.4 M (-1.3 MPa, isotonicity) to 1.0 M (-3.1 MPa, hypertonicity). There was a high degree of positive correlation between the extent of reduction in the area of detached leaf discs or the volume of protoplasts (indicated by diameter or absorption at 440 nm) and the decrease in photosynthesis. The correlation coefficients between inhibition of photosynthesis and the decrease in leaf disc area or protoplast volume were 0.96 and 0.99, respectively. We therefore suggest that the decrease in absorbance at 440 nm (corrected for turbidity at 750 nm) can be used as a simple measure to predict the inhibition due to osmotic stress of photosynthesis in mesophyll protoplasts. Similarly, the reduction in area of detached leaf discs could also be a very simple and useful criterion to assess osmotic tolerance of photosynthesis.     

Attachment force of the beetle Cryptolaemus montrouzieri (Coleoptera, Coccinellidae) on leaflet surfaces of mutants of the pea Pisum sativum (Fabaceae) with regular and reduced wax coverage

This study represents an investigation of surface-related plant–insect interactions. Surface micro-morphology of leaflets in pea (Pisum sativum) with wild-type crystalline surface waxes (waxy) and with reduced crystalline surface waxes (glossy) caused by a mutation (wel) were studied using various microscopy techniques. The free surface energy of these plant surfaces was estimated using contact angles of droplets of three different liquids. The morphological study of the attachment system in the ladybird beetle Cryptolaemus montrouzieri was combined with measurements of attachment (traction) forces, generated by beetles on these plant substrates. Differences were found in wax crystal shape, dimensions, and density between the adaxial and abaxial surfaces of waxy and glossy plants. The crystalline wax was not completely eliminated in the glossy plant: it was only slightly reduced on the adaxial side and underwent greater changes on the abaxial side. The free surface energy for both surfaces of both pea types was rather low with strongly predominating dispersion component. Insects generated low traction forces on all intact plant surfaces studied, except the abaxial surface of the glossy plant, on which the force was greater. After being treated with chloroform, all the surfaces allowed much higher traction forces. It is demonstrated that the difference in the crystal length and density of the epicuticular wax coverage within the observed range did not influence wettability of surfaces, but affected insect attachment. The reduction in insect attachment force on plant surfaces, covered with the crystalline wax, is explained by the decrease of the real contact area between setal tips of beetles and the substrate. Handling editor: Lars Chittka.     

Response of Pisum sativum (Fabales: Fabaceae) to Sitona lineatus (Coleoptera: Curculionidae) infestation: effect of adult weevil density on damage, larval population, and yield loss

Sitona lineatus L. (Coleoptera: Curculionidae) is an invasive pest in North America and its geographical range is currently expanding across the Canadian prairies. Adults and larvae of S. lineatus feed upon the foliage and root nodules, respectively, of field pea, Pisum sativum L. (Fabales: Fabaceae), and may contribute to economic losses when population densities are high. Integrated pest management (IPM) programs that incorporate economic thresholds should be used to manage S. lineatus populations in a sustainable manner. The impact of nitrogen fertilizer on field pea yield and the relationships between adult weevil density and above- and below-ground damage and yield were investigated in southern Alberta, Canada using exclusion cages on field pea plots. In each cage, 32 field pea plants were exposed to weevil densities ranging from zero to one adult weevil per plant. Nitrogen-fertilized plants yielded 16% more than unfertilized plants. Nitrogen-fertilized plants had fewer root nodules than unfertilized plants, but fertilizer had no effect on foliar feeding by S. lineatus. Adult density affected foliar feeding damage, with increases in above-ground damage associated with increases in S. lineatus density. Adult density did not affect root nodule damage, larval density, foliar biomass or seed weight. Overall, these results indicate that terminal leaf damage may be used to estimate adult weevil density but cannot be used to predict larval density or yield loss. Further research is required to better understand the impact of larval damage on yield and determine if economic thresholds can be developed using data from large-scale production systems.     

Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase.

Ursane type triterpene is one of the most widespread triterpene aglycones found in plants, together with oleanane type, and these two types often occur together in the same plant. Pisum sativum is known to produce both types of triterpenes. Homology based PCRs with degenerate primers designed from the conserved sequences found in the known beta-amyrin synthases have resulted in cloning of two triterpene synthase cDNAs from immature seeds of P. sativum. They show high sequence identities to each other (78%) and also to the known beta-amyrin synthases (70-90%). ORFs of the full-length clones named as PSY (2277 bp, codes for 759 amino acids) and PSM (2295 bp, codes for 765 amino acids) were ligated into the yeast expression vector pYES2 under the control of GAL1 promoter. Heterologous expression in yeast revealed PSY to be a P. sativum beta-amyrin synthase. Surprisingly, however, PSM turned out to be a novel mixed amyrin synthase producing both alpha- and beta-amyrin. Several minor triterpenes were also identified as the PSM byproducts. The presence of such multifunctional triterpene synthase would account for the co-occurence of ursane and oleanane type triterpenes in plants.     

Roles of the af and tl genes in pea leaf morphogenesis: characterization of the double mutant (afaftltl)

The pleiofila phenotype (afaftltl double mutant) of Pisum sativum arises from two single-gene, recessive mutations known to affect the identity of leaf pinnae, afila (af), and acacia (tl). The wild-type leaf consists of proximal leaflets and distal tendrils, whereas the pleiofila leaf consists of branched pinnae terminating in small leaflets. Using morphological measurements, histology, and SEM, we characterized the variation in leaf form along the plant axis, in leaflet anatomy, and in leaf development in embryonic, early postembryonic, and late postembryonic leaves of aftl and wild-type plants. Leaves on aftl plants increase in complexity more rapidly during shoot ontogeny than those on wild-type plants. Leaflets of aftl plants have identical histology to wild-type leaflets although they have smaller and fewer cells. Pinna initiation is acropetal in early postembryonic leaves of aftl plants and in all leaves of wild-type plants, whereas in late postembryonic leaves of aftl plants pinna initiation is bidirectional. Most phenotypic differences between these genotypes can be attributed to differential timing (heterochrony) of major developmental events.     

Interactions between Ethylene, CO(2), and ABA on GA(3)-Induced Amylase Synthesis in Barley Aleurone Tissue

Gibberellic acid-induced synthesis and release of α-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO₂ was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO₂ tested from 400 to 10⁵ microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases.     

Interactions over time between cereal leaf beetle (Coleoptera: Chrysomelidae) and larval parasitoid Tetrastichus julis (Hymenoptera: Eulophidae) in Utah

The phenology of parasitism of the cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae) by Tetrastichus julis (Walker) (Hymenoptera: Eulophidae) was studied in small grain fields from 2000 to 2005 in northern Utah, after release and redistribution of the partially bivoltine larval parasitoid during the 1990s. Host larvae first occurred in May, with peak infestation typically occurring in early to mid-June. Parasitism by overwintering females of T. julis was highest among earliest developing beetle larvae. Thereafter, rates of parasitism fell to low levels (5-10% or less) by the latter half of June, when heat accumulation had reached 280-350 degree-days (based on a minimum threshold of 8.9 degrees C). With the emergence of second generation parasitoids, rates of parasitism rose to levels approaching 100% among the relatively few late-developing larvae of O. inelanopus. Clear and consistent differences over the years were not observed among different crops of small grains (barley, wheat, or oats) either in the phenology and intensity of beetle infestation, or in the rate of parasitism of beetle larvae. The rate of parasitism was especially high in 2005, and an increase in the minimum level of parasitism (observed each year at mid-season) was apparent over the course of the study. These results indicate that the parasitoid has become well established and seems to be continuing to increase in its impact on O. melanopus in northern Utah, despite a relatively hostile environment of crop management, wherein most fields are plowed and disked annually.     

Enzymes of assimilatory sulfate reduction in leaves of Pisum sativum: Activity changes during ontogeny and in vivo regulation by H2S and cyst(e)ine

Enzyme activities of assimilatory sulfate reduction were measured in leaves of Pisum sativum L., cv. Vatters Frühbusch, during their ontogenetic development, and during treatment with H₂S and cyst(e)ine. Ribulose bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and ferredoxin-dependent nitrite reductase (Fd-NiR, EC 1.7.7.1) were measured for comparison. In etiolated pea leaves, ATP-sulfurylase (ATPase, EC 2.7.7.4), adenosine 5'-phosphosulfate sulfotransferase (APSSTase), ferredoxin-dependent sulfite reductase (Fd-SiR, EC 1.8.7.1) and O-acetyl-L-serine sulfhydrylase (OASSase, EC 4.2.99.8) activities were measured in appreciable rates, while neither RuBP carboxylase nor Fd-NiR activities could be detected.
During the first 2–7 days after transfer into the light all enzyme activities increased. After reaching maximal activities, ATPase, APSSTase, and Fd-SiR activities decreased in all leaves to low or indetectable levels during the following 3–6 days. RuBP carboxylase, Fd-NiR and OASSase, on the other hand, decreased slowly and were still at high levels of activity at the end of the experiment.
Fumigation of pea plants with 1.5 μl l⁻¹ H₂S delayed the initial increase and the subsequent decrease of ATPase activity by 1–3 days. APSSTase activity decreased for 1–2 days, increased rapidly during the next 4–6 days and retained a high level of activity until the end of the experiment as did Fd-SiR. One to two days after the beginning of fumigation the leaves started to accumulate high amounts of cyst(e)ine.
When pea plants with excised roots were placed on a nutrient solution containing cyst(e)ine, APSSTase activity decreased more on 0.2 and 0.5 m M than on 1.0 m M. Fd-SiR activity was only slightly decreased on 1.0 m M cyst(e)ine. Neither Fd-NiR nor RuBP carboxylase activities were affected.