Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA₁ or its immediate precursors. The primary site of synthesis of GA₂₀ from GA₁₉ was immature leaflets and tendrils, and the synthesis of bioactive GA₁ and its inactive catabolite GA₈ occurred predominantly in stem tissue. GA₂₉, the inactive catabolite of GA₂₀, was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA₁, GA₁₉, or even GA₂₀. During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [³H]GA₁ (and [³H]GA₂₉) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA₂₀ 3β-hydroxylase protein.     

Gibberellins in Light-Grown Shoots of Pisum sativum L. and the Influence of Reproductive Development

The presence of GA₉, GA₁₉ and GA₂₀ was demonstrated by gas chromatography/massspectrometry (GC/MS) and the presence of GA₄₄ strongly indicatedby GC/MS in selected ion monitoring mode (GC/SIM) in extractsof shoots of light-grown tall peas (Pisum sativum L.). Usingthe rice seedling bioassay with cv. Tan-ginbozu, the levelsof gibberellins in pea shoots were monitored from early shootgrowth through to apical senescence in a tall pea line. Levelsof activity corresponding to GA₂₀, GA₁₉ and GA₄₄ remained relativelystable in the shoot despite reproductive development and apicalsenescence. The level of GA₁-like activity increased to a maximumwhen the leaves had between 7 and 9 leaves expanded and decreasedonly with apical senescence. The na gene which blocks the productionof biologically active gibberellins in shoots but not in developingseed, was also operative in pod walls, with na pods containinglittle or no significant gibberellin-like activity when comparedto na pods at contact. This occurred despite the presence ofrelatively high levels of gibberellins in developing seed atthe same time. The results suggest that there is little or nosignificant leakage of biologically active gibberellins fromdeveloping seed to pods or shoots. Extracts of pods of tallpeas with Na contained low levels of gibberellin-like activitybut like developing seed, contained little or no significantGA₁-like activity despite the presence of significant GA₁-likeactivity in shoot extracts of tall peas. (Received March 11, 1986; Accepted May 27, 1986)     

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

Carbon Transfer and Partitioning between Vegetative and Reproductive Organs in Pisum sativum L

Assimilate partitioning was studied in the common pea (Pisum sativum L.) by feeding ¹⁴CO₂ to whole plants and measuring radioactivity in different organs 48 hours after labeling. Two experimental protocols were used. For the first, one reproductive node was darkened with an aluminum foil, to prevent photosynthesis during labeling. The aim was to study assimilate translocation among nodes. The second was carried out to assess any priority among sinks. Whole plants were shaded, during labeling, to reduce carbon assimilation. Various developmental stages between the onset of flowering and the final stage in seed abortion of the last pod were chosen for labeling. When all photosynthetic structures at the first reproductive node were darkened at any stage of development after the formation of the first flower, the first pod was supplied with assimilates from other nodes. In contrast, later developed pods, when photosynthetic structures at their node were darkened, received assimilates from other nodes only when they were beyond their final stage in seed abortion. Reducing illumination to 30% did not change distribution of assimilated carbon between vegetative and reproductive structures, nor among pods. It appears that the relative proportion of ¹⁴C allocated to any one pod, compared to other pods, depends on the dry weight of that pod as a proportion of the total reproductive dry weight. When the plant was growing actively, following the start of the reproductive phase until a few days before the end of flowering, the top of the plant (i.e., all the organs above the last opened flower) had a higher sink strength and a higher relative specific activity than pods, suggesting that it was a more competitive sink for assimilates. The pattern of assimilate distribution described here provides an explanation for pod and seed abortion.     

The Relationship of Endogenous Gibberellins to Light-regulated Stem Elongation Rates in Dwarf and Normal Cultivars of Pisum sativum L.

Dwarf cultivar Progress No. 9 and normal cultivar Alaska ofPisum sativum L. were grown under conditions of darkness orred light. Red light decreased the stem elongation rate of bothcultivars. Gibberellins present during the linear phase of stemelongation were isolated and the two main components were tentativelyidentified by gas chromato-chromatography and mass spectrometryas Gibberellin A₁ and A₅. Both gibberellins varied quantitativelywithin and between cultivar and treatment groups, and the amountspresent were inversely related to stem elongation rate. Althoughthe stem elongation rate of plants grown under red light wasrepressed, endogenous gibberellin was not limiting and was asmuch as two-fold higher in red light-grown plants than in dark-grownplants. The levels of endogenous gibberellin in dawrf plantsindicated that the genetic growth limitation was not due toa gibberellin deficiency. (Received May 26, 1981; Accepted January 28, 1982)     

Expression of the le Mutation in Young Ovaries of Pisum sativum and Its Effect on Fruit Development

The effect of the le mutation on the growth and gibberellin (GA) content of developing fruits was investigated using the near-isogenic lines of Pisum sativum L. 205+ (LeLe) and 205- (lele). Although stem elongation is known to be reduced in 205- plants by approximately 65%, the growth of pods and seeds was unaffected by the le mutation. GA1, GA3, and GA20 stimulated parthenocarpic development of unpollinated ovaries on both 205+ and 205- plants. GA20 was less active on 205- ovaries than on 205+, whereas GA1 had similar, high activity in both lines. The activity of GA3 was even higher than that of GA1 in both lines. Decapitation of 205+ plants induced parthenocarpic development of unpollinated ovaries, but this treatment was much less effective on 205- plants. The contents of GA1 and GA8 in entire ovaries 6 d after anthesis, as well as in the pod and fertilized ovules, were substantially lower in 205- than in 205+ plants, whereas the reverse was true for the levels of GA20 and GA29. These results suggest that 3[beta]-hydroxylation of GA20 to GA1 is reduced in ovaries as well as in vegetative tissues. Thus, the le mutation appears to be expressed in young reproductive organs of the 205- line, even though it does not affect the fruit phenotype. Because the content of GA3 in the ovary was similar in the two lines, one explanation for the normal fruit size in the 205- line is that GA3 is the native regulator of pod growth. Alternatively, sufficient GA1 may still be produced in 205- fruits to maintain normal pod growth.     

The Distribution of {beta}-Glycerophosphatase in Young Roots of Pisum sativum L.

The distribution of ß-glycerophosphatase activityin young roots of Pisum sativum, cultivar Alaska, has been examinedby biochemical and histochemical methods. Results obtained bythe two approaches are broadly similar, and indicate that highenzyme activity is associated with cells of the root cap, outerlayers of the cortex, differentiating xylem elements and phloemfibres, and cortical cells surrounding emerging lateral roots.The significance of this distribution in relation to a possiblefunction of ß-glycerophosphatase is discussed.     

The Translocation of 14C-photosynthate in Pisum sativum L.

Short-term studies were undertaken of source and sink relationshipsin Pisum sativum, cultivar Orfac, plants grown in a controlledenvironment. ¹⁴C-distribution assays were conducted after 24or 48 h ¹⁴Cphotoassimilate translocation from a single leafsubtending either a vegetative or a reproductive node. The primaryallocation of ¹⁴C-assimilate was achieved within 24 h: therewas no significant secondary movement of ¹⁴C within the subsequent24 h. The pod was strongly but not exclusively dependent onthe subtending leaf. Out of the total ¹⁴C fixed by a leaf theproportion that was exported within 24 h was related to the¹⁴C-sink capacity of the pods. A rapid non-combustive ¹⁴C-assay method is described whereby¹⁴C-tissue fragments are rendered translucent prior to directscintillation counting of the sample. In terms of cpm per mgobtained the latter method was comparable to a wet combustionmethod adapted for insoluble ¹⁴C-tissue.     

Metabolism of Phloem-borne Amino Acids in Maternal Tissues2Pisum sativum L.)

The amino acid composition of the EDTA-induced phloem exudatereaching the fruit and the seed, and of the solutes releasedby the seed coat during fruit development were determined inglasshouse-grown pea (Pisum sativum L. cv. Finale) suppliedeither with nitrate-free nutrients (nodulated plants) or withcomplete medium (non-nodulated plants). The EDTA-promoted exudationtechnique was used supposedly to collect phloem sap and theempty seed technique supposedly to collect the solutes secretedby the seed coat to the embryo sac cavity. In young seeds embryosac liquid was sampled directly from the embryo sac. The maincarbohydrate transported and secreted was sucrose. The mainamino acids reaching the fruit were asparagine, glutamine, andhomoserine. Their proportions were steady during a day-nightcycle and throughout fruit development. Amino acid compositionchanges occurred first in the pathway from fruit stalk to seedfunicle, due to the formation of threonine (probably from homoserine)and in the seed coat due to production of glutamine, alanineand valine which, together with threonine were the main secretedamino acids. The temporary nitrogen reserves of the pod walland seed coat were remobilized as asparagine during senescence.Phloem exudate of nodulated plants showed a higher (about twice)proportion of asparagine but lower proportions of homoserineand glutamine than in EDTA-induced phloem exudate of nitrate-fedplants. The two types of nitrogen nutrition also produced somechanges in relative proportions of threonine and homoserinesecreted by the seed coat. Key words: Pisum sativum, phloem, amino acids, pod wall, seed coat     

The control of damping-off in Pisum sativum L. by cycioheximide

Experiments on the effect of cycioheximide (Acti-dione) in controlling damping-off disease of peas confirmed that phytotoxicity would preclude its use despite its high toxicity to Pythium spp. The antibiotic did not inhibit germination of peas, but retarded growth of the shoot; the tap root was apparently unaffected, but the lateral roots were stunted.     

Carnitine acyltransferases in chloroplasts of Pisum sativum L.

Carnitine-acetyltransferase (EC 2.3.1.7) and carnitine-palmitoyltransferase (EC 2.3.1.21) activities were shown to be present in chloroplasts of green pea leaves and possibly to occur in leaf mitochondrial and peroxisomal fractions. A role for the enzymes in the transfer of acyl groups across membranes is suggested.     

Dominance,overdominance and epistasis in Pisum sativum L.

Summary Dominant genes are the main cause of the heterosis induced by fasciated mutants of different lines of Pisum sativum. Most of these cases were originally interpreted by different authors as examples of monogenic overdominance. Several not-closely-linked genes appear to have mutated simultaneously in most of the fasciated lines. Although fasciation itself is recessive, other mutant characters, such as lateness, increased stem length (number and length of internodes) and, in part, seed production per plant, show dominant inheritance. The latter two features are, however, to a considerable extent suppressed in the fasciated lines by unfavourable gene-interactions (epistasis). Crossing these lines with non-fasciated ones shows that the epistatic genes are recessive and the dominant genes are then no longer hindered in their action. By eliminating the epistatic genes from the genomes of fasciated lines by recombination, the heterosis phenomenon has been fixed on six independent occasions for different lines. The fasciata genes themselves were found to be the most probable cause of these cases of recessive epistasis. The question whether different kinds of fasciation affect heterosis differently is examined. Recessive epistasis and dominance explain most of the quantitative distinctions between the different hybrids. In addition, one example of heterosis between non-fasciated lines is given and the possible meaning of the overall results for plant breeding and population genetics is mentioned.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

The Effect of Fruit Shading on Yield in Pisum sativum L.

Fruits of Pisum sativum L. cv. Feltham First which initiatesonly one flower per flowering node, were selectively shadedunder varying levels of defoliation. The purpose of the experimentswas to ascertain whether the foliage could compensate for lossof the fruit's contribution to its own growth. There was evidenceof this, but fruit and seed weight per fruit and per plant werereduced by fruit shading at all levels of defoliation. The lossin yield due to shading suggested that the contribution fromthe fruit was at least 12 per cent. The number of seeds whichdeveloped to maturity was the yield component most affectedby treatment. There was no evidence to suggest that shadinghad a different quantitative effect on final weight at differentnodes, but it did increase flower abscission at the first foweringnode in an experiment done at low radiant exposure. In an experimentat higher radiant exposure, very few flowers abscised at theearlier nodes, but leaflet removal reduced final fruit yieldat the first flowering node to a greater degree than at thesecond. These differential responses could contribute to variabilityof seed size in a crop of vining peas. Pisum sativum, pea, fruit, pod, light, shading, photosynthesis, yield     

Stomata on the Primary Root of Pisum sativum L.

The first record of stomata on a non-specialized root was obtainedby scanning electron microscopy of 4-d-old Pisum sativum L.In some cases subsidiary cells were trichoblasts. Stomata andthe root triarch vascular structure were simultaneously presentin transverse sections through the root. Pisum sativum, pea, root stomata, guard cells, trichoblasts     

An Analysis of Seed Development in Pisum sativum L.

Growth analysis has been performed on developing seeds and seedcomponents of six contrasting genotypes of Pisum sativum. Seeddevelopment has been divided into three phases of high growthrate separated by two ‘lag’ phases, or phases oflow growth rate. It is suggested that the timing of these growthphases may not be determined by the developing seed, since thereappeared to be no consistent correlation with particular physiologicalstages of seed development. The relationship between the absolutegrowth rate of the embryo sac and of the embryo as determinedfrom changes in volume is reflected in the accumulation andabsorption of the endosperm. The relative growth rate of theembryo volume was invariably higher than that of the embryosac although the difference between these two relative ratesvaried with genotype and may account in part for the differencein seed phenotype. It is suggested that the testa and embryo are sinks which bothcompete for the endosperm which may act as a common source,and that this relationship accounts for variation in endospermvolume. It is concluded that seed development is dependent on threelevels of plant organization, the maternal parent, interactionsbetween the components of the seed and the genetic constitutionof the embryo. Pisum sativum L., garden pea, seed development, growth analysis     

An Acidic Polysaccharide in Seeds of Pisum sativum L.

A phenanthrene-assimilating bacterium which belongs to the genus Aeromonas was isolated from soil. The cells which adapted to phenanthrene required a growth lag time on a naphthalene medium. The cells oxidized l-hydroxy-2-naphthoate (1H2NA), 2-carboxybenzaldehyde (2CBAL), o-phthalate (OPA) and protocatechuate (PCA) but did not oxidize salicylaldehyde (SAL), salicylate (SA) and catechol (CAT) which are intermediates in naphthalene catabolism. Using the cell-free extract, the same results were obtained in oxidative capacity. The intact cells metabolized 1H2NA and 2CBAL without the lag time, giving 2CBAL and PCA, respectively. The ammonium sulfate-treated extract prepared from the cells grown in phenanthrene medium, converted 1H2NA to 2CBAL and 2CBAL to OPA. It was suggested that the Aeromonas sp. degraded phenanthrene through OPA.     

Functional Significance of Acid Phosphatase Distribution During Embryo Development in Pisum sativum L

The distribution of P₁, ester P and acid-insoluble nucleic acidP has been studied in relation to acid phosphatase activity(EC 3. 1. 3. 2) in the component parts of developing pea seeds(Pisum sativum L.). Despite the favourable pH of the liquidcontents of the embryo sac (pH 5.5), only very low acid phosphataseactivity was detected in this fluid (c. 0.01 units per seed).Potential substrates for phosphatase action were in fact absentfrom the secretion, the only form of P present being P_i, inconcentrations up to 8 mM. The data support the hypothesis thatthe high acid phosphatase activities which develop in the seed-coatsare involved in regulating the supply of P as P₁ to the developingembryo. Pisum sativum L., pea, embryo development, acid phosphatase, phosphorus, seed-coats, seed development