Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. I. Extent of transport through the coat symplast

The extent of post-phloem solute transport through the coatsymplasts of developing seeds of Vicia faba L. and Phaseolusvulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeantfluorochrome, CF, moved radially from the chalazal vein to reachthe chlorenchyma and thin-walled parenchyma transfer cell layers.Thereafter, the fluorochrome moved laterally in these two celllayers around the entire circumference of the seed coat. Transferof CF from the chalazal vein was inhibited by plasmolysis ofattached ‘empty’ seed coats. In contrast, the spreadof phloem imported CF was restricted to the ground parenchymaof Phaseolus seed coats. Fluorochrome loaded into the outermostground parenchyma cell layer was rendered immobile followingplasmolysis of excised seed-coat halves. Phloem-imported [¹⁴C]sucroseand the slowly membrane permeable sugar, L-[¹⁴C]glucose, werepartitioned identically between the vascular and non-vascularregions of intact Vicia seed coats. For ¹⁴C-photosynthates,these partitioning patterns in attached ‘empty’Vicia seed coats were unaffected by PCMBS, but inhibited byplasmolysis. Tissue autoradiographs of intact Phaseolus seedcoats demonstrated that a pulse of ¹⁴C-photosynthate moved fromthe veins to the grounds tissues. In excised Vicia seed coats,preloaded with ¹⁴C-photosynthates, the cellular distributionof residual ¹⁴C-label was unaffected by PCMBS. In contrast,PCMBS caused the ¹⁴C-photosynthate levels to be elevated inthe veins and ground parenchyma relative to the branch parenchymaof Phaseolusseed coat halves. Based on the above findings, itis concluded that the phloem of Vicia seed coats is interconnectedto two major symplastic domains; one comprises the chlorenchyma,the other the thin-walled parenchyma plus thin-walled parenchymatransfer cells. For Phaseolusseed coats, the phloem forms amajor symplastic domain with the ground parenchyma. Key words: Phaseolus vulgaris L, phloem unloading, photosynthate transport, seed coat, symplast, Vicia faba L     

Poplar Lignin Decomposition by Gram-Negative Aerobic Bacteria

Eleven gram-negative aerobic bacteria (Pseudomonadaceae and Neisseriaceae) out of 122 soil isolates were selected for their ability to assimilate poplar dioxane lignin without a cosubstrate. Dioxane lignin and milled wood lignin degradation rates ranged between 20 and 40% of initial content after 7 days in mineral medium, as determined by a loss of absorbance at 280 nm; 10 strains could degrade in situ lignin, as evidenced by the decrease of the acetyl bromide lignin content of microtome wood sections. No degradation of wood polysaccharides was detected. Lignin biodegradation by Pseudomonas 106 was confirmed by ¹⁴CO₂ release from labeled poplar wood, although in lower yields compared with results obtained through chemical analysis based on acetyl bromide residual lignin determination.     

The Role of the Oxygen Permeability of the Seed Coat in the Dormancy of Seed of Xanthium pennsylvanicum Wallr

A method for direct identification and quantitative measurementsof mixed or pure gases diffusing through seed coats was devisedto test the hypothesis that the dormancy of Xanthium pennsylvanicumseeds is caused by oxygen-impermeable seed coats. The diffusionof oxygen through seed coats of X. pennsylvanicum was shownto obey Fick's first law. Oxygen diffused through the lowerand upper seed coats at the same rate. Imbibed lower and upperseeds showed essentially equal oxygen uptake rates before radicleemergence. This uptake was lower than the rate at which oxygencan diffuse into the seed. Therefore upper seeds are not dormantbecause of seed coat restriction of oxygen diffusion. The relationshipsof oxygen with other factors involved in seed dormancy are discussed.     

Structure and Macromolecular Composition of the Seed Coat of the Musaceae

Mature seed coats of representatives of all three genera ofMusaceae were analysed for macromolecular composition with variousmass spectrometric techniques and compared with scanning electronmicroscopy and light microscopy in combination with histochemicaltechniques. Mass spectrometric techniques are more sensitiveand more specific in identifying macromolecular compounds thanhistochemical methods. The macromolecular ‘fingerprint’of the seed coats of Musaceae showed unique components of aromaticphenols. The seed coat structure of all three genera is homogeneouswithin the Musaceae. It is characteristic at the family leveland most complex within the Zingiberales. Very remarkable arethe separation of the outer cell walls from the exotestal layer,exposing a secondary surface with silica crystals, and the relativelythick mesotesta which protects the seed, e.g. against the bitingforces and passage through the digestive tracts of dispersingagents. Germination takes place with an operculum and is facilitatedby a predetermined rupture layer in the micropylar collar. Themusaceaous seed presents a good example of the solution of conflictingdemands of protection and germination. Musaceae; Musa; Ensete; Musella; seed coat; pyrolysis (gas chromatography) mass spectrometry; histochemistry; anatomy; macromolecules; silica; lignin; cellulose; vegetable polyphenols; operculum; germination     

The Acetyl Bromide Method Is Faster,Simpler and Presents Best Recovery of Lignin in Different Herbaceous Tissues than Klason and Thioglycolic Acid Methods

We compared the amount of lignin as determined by the three most traditional methods for lignin measurement in three tissues (sugarcane bagasse, soybean roots and soybean seed coat) contrasting for lignin amount and composition. Although all methods presented high reproducibility, major inconsistencies among them were found. The amount of lignin determined by thioglycolic acid method was severely lower than that provided by the other methods (up to 95%) in all tissues analyzed. Klason method was quite similar to acetyl bromide in tissues containing higher amounts of lignin, but presented lower recovery of lignin in the less lignified tissue. To investigate the causes of the inconsistencies observed, we determined the monomer composition of all plant materials, but found no correlation. We found that the low recovery of lignin presented by the thioglycolic acid method were due losses of lignin in the residues disposed throughout the procedures. The production of furfurals by acetyl bromide method does not explain the differences observed. The acetyl bromide method is the simplest and fastest among the methods evaluated presenting similar or best recovery of lignin in all the tissues assessed.     

Production of a Fungistat and the Role of Fungi during Germination of Digitalis purpurea L.cv. Gloxiniaflora Seeds

The seed coats of Digitalis purpurea L. cv. Gloxiniaflora werecultured for indigenous fungi. Alternaria alternata (Fries)was identified as the sole fungus on seeds freshly harvestedfrom unopened capsules, whereas A. alternata, Rhizopus arrhizus(Fischer), Penicillium sp. and other fungi appeared on storedseeds. The appearance of fungi in seed cultures was seasonal,being more frequent in winter and early spring than in summerand autumn. Alternaria grew well on autoclaved seeds, on dehiscentseed coats, or on seed coats separated from the embryos of ungerminatedseeds. Rhizopus did not grow on these but grew weakly on theculture medium from viable seeds. A. alternata appears to functionas a degradation agent for the seed coat subsequent to germination.Neither fungus was found to be essential to germination of Digitalisseeds. Bioassay of the culture medium from germinating seedsshowed that a fungistat effective against both Alternaria andRhizopus is produced coincident with germination. Based on chromatographicanalysis, the fungistat appears to be a cardenolide. Alternaria alternata (Fries), cardenolides, Digitalis purpurea L. cv, Gloxiniaflora, fungistats, germination, Rhizopus arrhizus (Fischer)     

Phloem Unloading within the Seed coat of Pisum sativum Observed using Surgically Modified Seeds

Phloem unloading in pea seed coats was observed by removingthe embryos from developing seeds and washing the attached coatswith a weakly buffered solution. The quantity of labelled photosynthateappearing in the washing solution varied immediately when thesolute concentration was changed, and is shown to be an osmoticresponse. This response is predicted by the Münch theoryof phloem transport with concentration dependent unloading.Respiratory inhibitors and the sulphydryl modifying reagentPCMBS had a slow effect upon the washout of tracer, which arrivedwithin the seed coat prior to inhibitor application, but completelystopped any washout of tracer arriving after its application.This time-course suggests that the inhibitors were not directlyinhibiting unloading, but preventing further tracer from enteringthe region of unloading within the seed coat. Phloem unloadingwithin the seed coats of Pisum appears to be passive and notdependent upon a PCMBS-sensitive carrier. Key words: Pisum sativum, seeds, phloem unloading     

The Influence of Genotype, Temperature and Moisture on Seed Longevity in Chickpea, Cowpea and Soya bean

Seed of three chickpea (Cicer arietinum L.), three cowpea [Vignaunguiculata (L.) Walp.] and four soya bean [Glycine max (L.)Merr.] cultivars were hermetically stored for up to 2 yearsin various constant environments which included temperaturesfrom —20 to 70 °C and moisture contents (fresh weightbasis) from 5 to 25 per cent. In all cases the survival curvescould be described by negative cumulative normal distributions.The longevity of the various seed lots differed but the valueof the standard deviation (the reciprocal of which gives theslope of the survival curve when percentage germination is transformedto probit) was the same for all cultivars within a species whenstored under similar conditions. Within each species the relativeeffects of moisture and temperature on longevity did not differsignificantly between cultivars. In all three species therewas a negative logarithmic relationship between seed moisturecontent and longevity, but the relative effect of moisture contentdiffered between the species: differences in the longevity ofsoya bean seed as a function of moisture content were less thanfor either cowpea or chickpea. The relative effect of temperatureon seed longevity did not differ between the three species,and the seed of all three species showed increasing temperaturecoefficients for the change in rate of loss of viability withincrease in temperature. The complete pattern of loss in viabilityin all three species can be described by a single equation whichwas developed for barley and has also been shown to apply toonion seed. The constants applicable to the three grain legumeshave been calculated so that it is now possible to predict percentageviability of any seed lot of these species after any storageperiod under a very wide range of storage conditions. Cicer arietinum L., chickpea, Glycine max (L.) Merr., soya bean, Vigna unguiculata (L.) Walp., cowpea, seed longevity, seed storage, moisture content, temperature     

Changes in Photosynthate Unloading from Perfused Seed Coats of Phaseolus vulgaris L. Induced by Osmoticum and Ethylenediaminetetraacetate (EDTA)

Photosynthate unloading in Phaseolus vulgaris L. seed coatswas studied by treating perfused seed coats with differing concentrationsof an osmoticum and ethylenediaminetetraacetate (EDTA). Largechanges in osmoticum concentration typically produced rapidchanges in efflux of unlabelled sugar and steady-state-labelled¹⁴C-photosynthate. Osmoticum-induced changes in photosynthateefflux were caused by phloem import stimulation at low cellturgor and net efflux stimulation by high cell turgor. Eventhough rapid changes in sugar and tracer efflux were often inducedby osmoticum treatments, the specific activity of sugar releasedfrom seed coats was not greatly affected by these treatmentsand was similar to the specific activity of sugar remainingin the seed coat after perfusion. Thus, tracer was transportedfrom the phloem throughout the seed coat sugar pool before itwas released to the apoplast. This result is most consistentwith symplastic phloem unloading throughout perfused seed coats,because apoplastic transport between cells within the seed coatwas blocked by perfusion. Photosynthate efflux was stimulatedby simultaneous treatment of seed coats with EDTA and differentconcentrations of an osmoticum; loss of photosynthate from seedcoats did not appear to be tissue-specific. Key words: Phaseolus vulgaris, seed coat, photosynthate unloading, turgor, EDTA     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase‐like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events.     

Coexistence but Independent Biosynthesis of Catechyl and Guaiacyl/Syringyl Lignin Polymers in Seed Coats

Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.     

Regulation of Leaf Senescence by Reproductive Sink Intensity in Cowpea (Vigna unguiculata L. Walp)

The relationship between seed number per pod and senescenceof the leaf in its axil was examined in a determinate cowpea(Vigna unguiculata L. Walp) variety C.779. The seed number perpod was reduced at all fruiting nodes by surgical excision ofpart of the 4-d-old pod. Leaf senescence as measured by lossof leaf area, chlorophyll content and soluble protein was sloweddown in leaves supporting the development of an artificiallyreduced number of seeds. Diminished nitrogen mobilization fromthe leaf could not account for the reduced rate of leaf senescence.The result suggests the involvement of a senescence signal fromthe developing seeds to the leaf in its axil. Development ofthe basal half of the excised pod in the cowpea provides a uniquesystem for manipulating seed number per pod. Senescence, monocarpic, chlorophyll, protein, Vigna unguiculata, cowpea     

Mechanisms of solute efflux from seed coats: whole-cell K+ currents in transfer cell protoplasts derived from coats of developing seeds of Vicia faba L.

In developing seed ofVicia faba L., solutes imported throughthe phloem of the coats move symplastically from the sieve elementsto a specialized set of cells (the thin-walled parenchyma transfercells) for release to the seed apoplast. Potassium (K⁺) is thepredominant cation released from the seed coats. To elucidatethe mechanisms of K⁺ efflux from seed coat to seed apoplast,whole-cell currents across the plasma membranes of protoplastsof thin-walled parenchyma transfer cells were measured usingthe whole-cell patch-clamp technique. Membrane depolarizationelicited a time-dependent and an instantaneous outward current.The reversal potential (E_R of the time-dependent outward currentwas close to the potassium equilibrium potential (E_K and itshifted in the same direction as E_K upon changing the externalK⁺ concentration, indicating that this current was largely carriedby an efflux of K⁺. The activation of the time-dependent outwardK⁺ current could be well fitted by two exponential componentsplus a constant. The instantaneous outward current could alsobe carried by K⁺ efflux as suggested by ion substitution experiments.These K⁺ outward rectifier currents elicited by membrane depolarizationare probably too small to represent the mechanism for the normalK⁺ efflux from seed coat cells. Membrane hyperpolarization morenegative than –80 mV activated a time-dependent inwardcurrent. K⁺ influx was responsible for the inward current asthe current reversed at membrane voltage close to E_K and shiftedin the same direction as E_K when external [K⁺] was varied. Activationof this K⁺inward rectifier current was well fitted with twoexponential components plus a constant. A regulating functionfor this current is suggested. Key words: Potassium outward rectifier, potassium inward rectifier, transfer cell protoplast, seed coat, Vicia faba L     

Expression of Cell Wall Proteins in Seeds and During Early Seedling Growth of Araucaria araucana is a Response to Wound Stress and is Developmentally Regulated

Araucaria araucana seeds and seedlings respond to wounding after48 h with a 3- to 4-fold increase of hydroxyproline-rich glycoproteins(HRGP) in the cell walls of the embryo and with a 15-fold increasein the cell walls of the megagametophyte. The megagametophytewalls accumulate six times more hydroxyproline per µgof cell wall protein than the embryo in this wound response.Tissue immunoprints of different parts of seeds and seedlingsobtained with polyclonal antibodies raised against HRGP fromcarrot roots or soybean seed coats indicate that the responseis due to an increase in a protein similar to the ones seenin carrot roots or soybean seed coats. Western blots of embryoand megagametophyte cell wall proteins subjected to SDS-PAGEshow three bands that cross-react with these antibodies. Ina native cationic gel system followed by Western blot analysis,only two bands react with these antibodies. Expression of suchproteins in Araucaria araucana seeds seems to be developmentallyregulated and tissue specific, since they are present mainlyin the megagametophyte and the root cap of the embryo. Key words: Araucaria araucana, seeds, seedlings, cell walls, hydroxyproline-rich glycoproteins     

Identification of candidate genes and molecular markers for heat-induced brown discoloration of seed coats in cowpea [Vigna unguiculata (L.) Walp]

Background

Heat-induced browning (Hbs) of seed coats is caused by high temperatures which discolors the seed coats of many legumes, affecting the visual appearance and quality of seeds. The genetic determinants underlying Hbs in cowpea are unknown.

Results

We identified three QTL associated with the heat-induced browning of seed coats trait, Hbs-1, Hbs-2 and Hbs-3, using cowpea RIL populations IT93K-503-1 (Hbs positive) x CB46 (hbs negative) and IT84S-2246 (Hbs positive) x TVu14676 (hbs negative). Hbs-1 was identified in both populations, accounting for 28.3% -77.3% of the phenotypic variation. SNP markers 1_0032 and 1_1128 co-segregated with the trait. Within the syntenic regions of Hbs-1 in soybean, Medicago and common bean, several ethylene forming enzymes, ethylene responsive element binding factors and an ACC oxidase 2 were observed. Hbs-1 was identified in a BAC clone in contig 217 of the cowpea physical map, where ethylene forming enzymes were present. Hbs-2 was identified in the IT93K-503-1 x CB46 population and accounted for of 9.5 to 12.3% of the phenotypic variance. Hbs-3 was identified in the IT84S-2246 x TVu14676 population and accounted for 6.2 to 6.8% of the phenotypic variance. SNP marker 1_0640 co-segregated with the heat-induced browning phenotype. Hbs-3 was positioned on BAC clones in contig512 of the cowpea physical map, where several ACC synthase 1 genes were present.

Conclusion

The identification of loci determining heat-induced browning of seed coats and co-segregating molecular markers will enable transfer of hbs alleles into cowpea varieties, contributing to higher quality seeds.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-328) contains supplementary material, which is available to authorized users.     

Mechanism of Photosynthate Efflux from Vicia faba L. Seed Coats

In order to develop a tentative model of the mechanism of photosynthateefflux from the vascular region of Vicia faba L. seed coats,wash-out experiments were performed after removal of the embryo. The sulphydryl group modifiers, pCMBS and NEM, reduced ¹⁴C-photosynthateefflux by 40% and 50%, respectively. Their inhibitory effectcould be prevented or reduced (in the latter case) by includingDTT in the bathing solution. Maltose competed with sucrose forefflux; a concentration of 300 mol m^–3 inhibited ₁₄C-photosynthaterelease by 35%. The cations K⁺ , Na⁺ Mg²⁺ and TPP⁺ enhancedefflux significantly, whereas the countenon Cl^– had noeffect. The presence of the protonophore CCCP (0·1 molm^–3) led to a reduction of efflux by 50% net proton extrusiondropped by 34%. To a lesser extent, an efflux inhibition wasalso achieved by decreasing the cytoplasmic pH with the weakacid DM0. In contrast, alterations in the external pH causedonly a feeble response. The ATPase inhibitor, EB, decreasedphotosynthate efflux and H⁺ extrusion. DES reduced efflux slightly,presumably by affecting ATPase activity as well as energy metabolism. Based on these findings, it is proposed that a sucrose/protonantiport mechanism could be responsible for photosynthate effluxfrom Vicia faba seed coats. Key words: Photosynthate efflux, proton extrusion, proton/sucrose antiport, seed coat, Vicia faba L.     

The Importance of Genetically Determined Seed Coat Characteristics to Seed Quality in Grain Legumes

Comparisons of five pairs of isogenk lines of peas, differingonly in the A gene for seed coat colour showed that white seeds(genotype aa) imbibed more rapidly than coloured seeds (AA),suffered greater imbibition damage revealed by dead tissue onthe cotyledons, and higher solute leakage. Seed-coat pigmentationwas closely associated with slow water uptake, since when expressionof the A gene was suppressed by the recessive pollens gene,the resulting white seeds {palpal AA) imbibed rapidly. The slowwater uptake by coloured seeds was not due to the restrictionof water entry by the seed coat since the differences in imbibitionrate were maintained when a portion of the seed coat was removedand seeds were imbibed with the exposed cotyledon in contactwith moist filter paper. Imbibition of similarly treated seedsby immersion in polyethylene glycol solutions (1–4%) whichincreased the seed/solution wettability, had little effect onthe water uptake of coloured seeds compared to imbibition inwater whereas that of white seeds increased in the first 10mins imbibition. Poor wettability of the inner surface of colouredseed coats did not therefore explain the slow imbibition ofthese seeds. The white seed coats loosened rapidly during imbibitionwhilst the coloured seed coats remained closely associated withthe cotyledons suggesting that the adherence of the seed coatto the cotyledons and therefore the ease of access of waterbetween the testa and cotyledons determines the rate of imbibition.The rapid water uptake by white-coated seeds and the subsequentimbibition damage may explain the high incidence of infectionof these seeds by the soil-bome fungus Pythhan after 2 d insoil. Improved seed quality and emergence may therefore be achievedby breeding for seed coat characteristics leading to reducedrates of imbibition Pisum sativum, isogenic lines, A gene, seed coat colour, imbibition, imbibition damage, wettability, pollens gene, seed quality, grain legumes