First report of non-mycorrhizal plant mutants (Myc) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc⁻, are characterized by aborted infections limited to one or two host cells. Expression of the myc⁻ character is associated with that of the nod⁻ character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc⁻ mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.     

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.     

Functional organization of thylakoid membranes in viable pea mutants with low chlorophyll content

The functional organizations of thylakoid membranes from wild type pea ( Pisum sativum L. cv. Kapital) and two viable mutants with low chlorophyll (Chl) contents were compared. Nuclear mutations in mutants 7 and 42 led to two- and three-fold decrease in total chlorophyll content, respectively. In spite of low Chl content mutants showed 80% photosynthetic activity, biological productivity, and seed production. It has been shown that mutant membranes differed from that of wild type by Chl distribution between the pigment-protein complexes and by stoichiometry of the main electrontransport complexes. The ratio photosystem I (PSI): photosystem II (PSII): cytochrome (Cyt) bjf complex: Chl was 1:1.1:1.2:650 in wild type chloroplasts, 1:1.8:1.7:600 in mutant 7 , and 1:1.5:1.9:350 in mutant 42 . PSI- and PSII-dependent electron-transport activities were enhanced in the mutants per mg Chl in proportion to number of reaction centers. The activity of the non-cyclic electron-transport chain increased in proportion to PSII and Cyt bjf complexes. The amount of ATP synthetase per unit of Chl as estimated by H⁻ATPase activity was much greater in mutant thylakoids, which is favorable for photosynthetic energy transduction. The low content of the light-harvesting complexes (LHC) in mutants is compensated by an increase of the number of PSII and Cyt bjf complexes, which eliminates the bottleneck at the site of plastoquinone oxidation.     

Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation

Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod⁻), or with ineffective nodules (nod⁺, fix⁻) were obtained, whilst in the other 4 hypernodulated mutants (nod⁺⁺) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod⁻), (nod⁺, fix⁻) and (nod⁺⁺, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.

Grafting experiments showed the (nod⁻) and (nod⁺, fix⁻) phenotypes are associated with the root genotypes and that (nod⁺⁺, nts) phenotype is associated with the shoot genotype.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

ABA content in shoots and roots of pea mutants af and tl as related to their growth and morphogenesis

The comparative study of shoot and root growth was carried out, and the level of ABA therein determined in the mutant af and tl and wild-type isogenic lines of pea. The recessive af mutation transformed the leaflets into tendrils, and the tl mutation transformed the tendrils into leaflets. These mutations did not affect the length and number of internodes. In all plants, the level of ABA in the leaves was 3–10 times greater than in the roots, and in the course of vegetative growth it rose in both organs. An increase in the shoot area of tl mutant did not change the dry weight of underground and above-ground parts; therefore, the ratio shoot/root in the mutant was identical to that in the wild-type plants. The maintenance of shoot dry weight in the tl mutant at the level of wild-type plant while its area considerably increased was accounted for by a decrease in the thickness of the leaflet and stipule blades. The level of ABA in the stipules of mutant plants was greater than in the wild-type plants. A decrease in the shoot area in the af mutant brought about a decline in its dry weight; however, the ratio root/shoot was maintained at the wild-type level due to a reduced accumulation of dry weight by the root. The level of ABA in the roots of the af mutant was twice greater than in the leafy forms. ABA was assumed to participate in the control over the root growth exerted by the shoot. The absence of leaflets in the af plants was partially compensated for by expanding stipules. The level of ABA therein was three times higher than in the plants of wild type and comparable with the level in the leaflets of the tl mutant and in the wild-type plants. The role of ABA in the growth and final size of leaf blades is discussed.     

Interaction of 4-chloroindole-3-acetic acid and gibberellins in early pea fruit development

In pea, normal pod (pericarp) growth requires the presence of seeds; and in the absence of seeds, gibberellins (GAs) and/or auxins can stimulate pericarp growth. To further characterize the function of naturally occurring pea GAs and the auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), on pea fruit development, profiles of the biological activities of GA3, GA1, and 4-Cl-IAA on pericarp growth were determined separately and in combination on pollinated deseeded ovaries (split-pericarp assay) and nonpollinated ovaries. Nonpollinated ovaries (pericarps) responded differently to exogenous GAs and 4-Cl-IAA than pollinated deseeded pericarps. In nonpollinated pericarps, both GA3 and 4-Cl-IAA stimulated pericarp growth, but GA3 was significantly more active in stimulating all measured parameters of pericarp growth than 4-Cl-IAA. 4-Cl-IAA, GA1, and GA3 were observed to stimulate pericarp growth similarly in pollinated deseeded pericarps. In addition, the synergistic effect of simultaneous application of 4-Cl-IAA and GAs on pollinated deseeded pericarp growth supports the hypothesis that GAs and 4-Cl-IAA are involved in the growth and development of pollinated ovaries.     

Population genetic structure and classification of cultivated and wild pea (Pisum sp.) based on morphological traits and SSR markers

Pea (Pisum sativum L.) is an important legume crop that is widely grown worldwide for human consumption and livestock feed. Despite extensive studies, the population genetic structure and classification of cultivated and wild pea (Pisum sp.) remain controversial. To characterize patterns of genetic and morphological variation and investigate the classification of Pisum, we conducted comprehensive population genetic analyses for 323 accessions from cultivated and wild pea, representing three species of Pisum and utilizing 34 morphological traits and 87 polymorphic simple sequence repeat markers. First, we identified three distinct genetic groups among all samples. Group I was primarily composed of Pisum fulvum, Pisum abyssinicum, and some wild P. sativum accessions, whereas Groups II and III consisted of the two genetic groups under P. sativum that represented different geographic distributions of cultivated pea. Analyses of morphological variation revealed significant differences among the three species. Second, among pea germplasms representing eight taxa of Pisum, P. fulvum and P. abyssinicum possessed unique genetic backgrounds and morphological characteristics, corroborating their independent species status. The intraspecific subdivisions of P. sativum described by some authors were not supported in this study, with the exception of several genotypes of P. sativum subsp. elatius that clustered with P. fulvum and P. abyssinicum. Finally, we confirmed that the Chinese pea germplasm was genetically distinct and could be divided into two genetic groups, each of which included both spring-sowing and autumn-sowing ecotypes. These results provide a robust foundation for understanding pea domestication and the utilization of wild genetic resources of pea.     

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.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

豌豆种子萌发时的含水量对子叶中蛋白酶和淀粉酶活性的影响

不同含水量的豌豆种子在饱和水蒸气中保持7d,在此过程中,当含水量低于萌动临界含水量时,限制了子叶中淀粉酶长寿mRNA的转译;含水量达到或超过萌动临界含水量时,子叶中淀粉酶长寿mRNA的转译得以进行,但转译程度因含水量不同而异,正常生长的轴器官是吸收子叶中蛋白质和淀粉水解产物的动力库,子叶中氨基酸含量的变化灵敏地调节着蛋白酶的活性,从而影响着蛋白质的动员程度,当环境中供水不足时,发现子叶中还原糖积累很快,非还原糖含量几乎没有变化,因此,认为对淀粉酶活性起灵敏调节作用的主要是还原糖。     

孟德尔豌豆基因克隆的研究进展及其在遗传学教学中的应用

150多年前, 孟德尔进行了豌豆7对相对性状的杂交试验, 发现了遗传学的两个基本规律。1900年, 孟德尔定律被重新发现以后, 人们从生理生化、细胞和分子水平等不同层次上对豌豆的这7个性状进行了深入研究。近年, 随着分子生物学技术的发展, 已有种子形状(R)、茎的长度(Le)、子叶颜色(I)和花的颜色(A)等4个性状的基因被克隆; 未成熟豆荚的颜色(Gp)、花的着生位置(Fa)和豆荚形状(V)的基因已被定位在各自的连锁群上。4个孟德尔基因的鉴定和克隆加深了人们对基因概念的理解：如基因功能的多样性、在分子水平上基因变异原因的多样性、显性和隐性的分子实质等。在遗传学教学中, 把孟德尔基因克隆和研究的最新进展介绍给学生, 在分子水平上诠释经典遗传规律, 有助于提高学生的学习兴趣, 帮助学生全面把握从形式遗传学到分子遗传学的内容和遗传学的发展方向。     

Two isoforms of the GBSSI class of granule-bound starch synthase are differentially expressed in the pea plant (Pisum sativum L.)

In addition to the GBSSI isoform of starch synthase described previously, the pea plant contains a second, granule-bound isoform, GBSSIb. GBSSI is abundant in pea embryos and Rhizobium root nodules, is present at low levels in pods and is absent from leaves. Mutations at the lam locus eliminate GBSSI from all of these organs. GBSSIb is present in pods, leaves and nodules and is unaffected by mutations at the lam locus. GBSSI and GBSSIb are very similar in molecular mass, primary sequence, activity and antigenic properties. GBSSIb, like GBSSI, can synthesize amylose in the presence of malto-oligosaccharides in isolated starch granules. However, its role in vivo is unclear. The lam mutation eliminates amylose from the starch of embryos but does not affect the relatively small amounts of amylose-like material in the starch of pods, leaves and nodules. The significance of these results for understanding of the regulation of amylose synthesis is discussed.     

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod⁻, Myc⁺). Received: 3 March 1997 / Accepted: 12 May 1997     

Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.