Formation of the vascular system of developing bean (Phaseolus limensis L.) seeds according to nuclear magnetic resonance microtomography

1H magnetic resonance microtomography imaging was applied to study vascular systems in developing bean (Phaseolus limensis L.) seeds. Using the gradient echo method, we recorded 2D tomographic sections in the sagittal and axial planes of the fruits sampled from a vegetating plant on days 10, 17, 24, and 31 after fertilization. Any vascular connection between the tissues of maternal plant (bean pod and seed coat) and the embryo were undetectable. The embryo has an autonomous branched network of procambial strands in the cotyledons, converging to the embryonic axis. The bean pods are covered with a network of vascular bundles; large vascular strands run along the dorsal and ventral sutures. The seed coat vascular bundles are formed in the process of seed ripening and are represented by a developed vascular system multiply branching in the middle part of the ground parenchyma at the stage of physiological maturity. They are connected with the source of assimilates via the lateral pod veins and a large vascular bundle, entering the seed below the hilum via the placenta. Assimilates enter the external part of the seed coat, which contains no vascular bundles, via the funiculus vascular bundles and hilum tissue.     

Vascular Bundle Distribution Within Broad Bean Seed Coat and the Structure of Unloading Region

Cytological observations were made on developing seed coat of broad bean (Vicia faba L.) by use of light and electron microscopy. Attention was focused on vascular distribution. The seeds were attached by the funiculus to tile large vascular bundles of pericarp of broad bean. The vascular bundle passed through hilum and two layers of palisade, entered the pa- renchyma of seed coat, then diverged in to two routes. One was a complete vascular bundle composed of both'phloem and xylem elements, it stretched down through seed raphe, then upward and terminated near the radical. The other was a two-recurrent-vascular-bundle with only phloem constitutents, they extended forward detoured the micropyle and extended downward, but did not join with the upward complete vascular bundle. The recurrent vascular bundles branched out many small short branches. The obvious difference between phloem of recurrent vascular bundle and of complete vascular bundle was that the companion ceils of the former did not normally modify to transfer ceils, but connected to the adjoining parenchyma cells through abundant plasmodesmata. It is deduced from the structural analysis that the symplastic route may play an important role, particularly in the region of recurrent vascular bundle, in the course of importing assimilates unloading in seed coat and transporting to the embryo.     

蚕豆籽粒内的~(14)C同化物的运转和卸出动态

通过向蚕豆叶片饲喂~(14)CO_2,应用液闪和显微放射性自显影技术表明标记同化物经叶脉和果荚韧皮部筛管快速运输至蚕豆种皮。种皮吸收营养、生长,后期逐步降解、供养子叶。种皮内的两类维管束系统同时输送营养并卸出到种皮内侧的质外体空间里。种皮里的反向维管束韧皮部卸出以共质体方式为主。并提供养分供种皮生长,而大部分的同化物由正向完整维管束韧皮部的筛分子一传递细胞进行质外体方式卸出。膨大中的子叶在早期即已成为生理上十分活跃的库。它对标记同化物的摄入随时间进程而急剧上升。     

Seed coat cell turgor in chickpea is independent of changes in plant and pod water potential

Turgor pressure in cells of the pod wall and the seed coat of chickpea (Cicer arietinum L.) were measured directly with a pressure probe on intact plants under initially dry soil conditions, and after the plants were irrigated. The turgor pressure in cells of the pod wall was initially 0.25 MPa, and began to increase within a few minutes of irrigation. By 2-4 h after irrigation, pod wall cell turgor had increased to 0.97 MPa. This increase in turgor was matched closely by increases in the total water potential of both the pod and the stem, as measured by a pressure chamber. However, turgor pressure in cells of the seed coat was relatively low (0.10 MPa) and was essentially unchanged up to 24 h after irrigation (0.13 MPa). These data demonstrate that water exchange is relatively efficient throughout most of the plant body, but not between the pod and the seed. Since both the pod and the seed coat are vascularized tissues of maternal origin, this indicates that at least for chickpea, isolation of the water relations of the embryo from the maternal plant does not depend on the absence of vascular or symplastic connections between the embryo and the maternal plant.     

Seed coat cell turgor responds rapidly to air humidity in chickpea and faba bean

The turgor pressure in cells of chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) seed coats was measured with a pressure probe. Measurements were made under in situ conditions by removing a section of wall from a pod, which remained attached to the plant, and exposing the intact seed. If the pod wall was removed and the turgor measurements made under ambient laboratory conditions of 50% to 70% relative humidity (RH), cell turgor pressure declined over time, typically reaching 0 MPa. If the pod wall was removed and the turgor measurements made under conditions of 100% RH, however, cell turgor pressure was stable over time, relatively uniform within the seed coat tissue, and was found to be 0.1-0.3 MPa for chickpea, and 0.1-0.2 MPa for faba bean. In both species there was a marked decline in cell turgor, beginning within about 60 s, when humidification was discontinued. The decline in cell turgor occurred regardless of the depth of the cell within the seed coat tissue, and this decline could be stopped, but not entirely reversed, when humidification was restored. An increase in cell turgor could also be caused by wetting of the seed. These responses indicate that a very rapid water exchange can occur within the seed coat tissue in situ. The rapid and, in some cases, relatively permanent loss of seed coat cell turgor in the absence of humidification raises serious concerns regarding desiccation artefacts which may be involved in the empty seed coat technique, often used to study seed carbon and water relations in grain legumes.     

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

黄精种子萌发过程发育解剖学研究

采用石蜡切片技术对成熟黄精种子形态及萌发过程中的形态学变化及解剖结构特征进行了研究,以阐明黄精种子繁殖的生物学机制。结果显示:(1)成熟的黄精种子由外而内依次为种皮、胚乳和胚等3部分组成。其中种皮由一层木质化的细胞组成;胚乳占据种子的大部分结构,胚乳细胞含有大量淀粉,细胞壁增厚;胚处于棒型胚阶段。(2)黄精种子在萌发过程中棒型胚靠近种脐端分化为吸器、子叶联结和子叶鞘,靠近种孔的部位分化出胚根、胚轴和胚芽。(3)黄精种子萌发首先由子叶联结伸长将胚芽和胚根原基推出种孔,紧接着下胚轴膨大形成初生小根茎,吸器留在种子中分解吸收胚乳中的营养物质。(4)通过子叶联结连通吸器和初生小根茎,将胚乳中的营养物质由吸器-子叶联结这个通路转移到初生小根茎中,为初生根茎上胚芽和胚根的进一步分化提供物质保障。(5)黄精种子自然条件下萌发率较低,而且当年不出土。研究表明,黄精种子的繁殖生物学特性是其生态适应的一种重要机制。     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Mobilization of Minerals to Developing Seeds of Legumes

The mineral nutrition of fruiting plants of Pisum sativum L.,Lupinus albus L. and Lupinus angustifolius L. is examined insand cultures supplying adequate and balanced amounts of essentialnutrients. Changes in content of specific minerals in leaves,pods, seed coat, and embryo are described. P, N and Zn tendto increase precociously in an organ relative to dry matteraccumulation, other elements more or less parallel with (K,Mn, Cu, Mg and Fe) or significantly behind (Ca and Na) dry weightincrease. Some 60–90 per cent of the N, P and K is lostfrom the leaf, pod and seed coat during senescence, versus 20–60per cent of the Mg, Zn, Mn, Fe and Cu and less than 20 per centof the Na and Ca. Mobilization returns from pods are estimatedto provide 4–39 per cent of the seeds' accumulations ofspecific minerals, compared with 4–27 per cent for testatransfer to the embryo. Endosperm minerals are of only minorsignificance in embryo nutrition. Comparisons of the mineral balance of plant parts of Lupinusspp. with that of stem xylem sap and fruit tip phloem sap supportthe view that leaves and pod are principal recipients of xylem-borneminerals and that export from these organs via phloem is themajor source of minerals to the seeds. Endosperm and embryodiffer substantially in mineral compostition from phloem sap,suggesting that selective uptake occurs from the translocationstream during seed development. Considerable differences are observed between species in mineralcomposition of plant organs and in the effectiveness of transferof specific minerals to the seeds Differences between speciesrelate principally to Ca, Na and certain trace elements.     

苏铁种子的种皮结构特征研究

对苏铁(Cycas revoluta Thunb.)种子的种皮进行了解剖研究,结果表明:苏铁种子的种皮分为外种皮、中种皮和内种皮3层结构.外种皮含有角质化的表皮细胞、薄壁细胞以及少量的厚壁细胞和异细胞,布有树脂道、气室和4束大维管束;中种皮主要由厚壁细胞群和木质化纤维组成,种孔端有一条缝合线,种脐端有3个孔;内种皮由多层干瘪的薄壁细胞和脉络状维管束组成,种孔端有一层椭圆状保护膜.对外种皮和内种皮维管束进行观察研究发现:外种皮和内种皮的维管束分布方式及其结构存在明显差异,外种皮的维管束由种脐端顺着种子弧形走向种孔端,内种皮的维管束呈脉络状,形成维管网贯穿其中;内、外种皮维管束中均存在多种不同样式的导管.     

Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits

Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Pathway of Photosynthate Transfer in the Developing Seed of Vicia faba L. Transfer in Relation to Seed Anatomy

The seed coat vascular system of the developing seed of Viciafaba consists of a chalazal and two lateral veins. The veinsare embedded in parenchymatous tissue which lies beneath thehypodermis and is divided into chlorenchyma, ground parenchymaand thin-walled parenchyma. The thin-walled parenchyma cellsand, in old seed coats, the vascular parenchyma of the veinsundergo additional secondary wall development to form transfercells. Thus, transfer cells line the entire inner surface ofthe seed coat. Initial distribution of ¹⁴C-photosynthates andsodium fluorescein within the seed coat was in the vascularsystem. Subsequent transfer towards the embryo was either radiallythrough vascular parenchyma and thin-walled parenchyma to thin-walledparenchyma/transfer cells, or by lateral spread within the groundand thin-walled parenchyma/transfer cells of the non-vascularregion of the seed coat prior to radial transfer. One-thirdof the ¹⁴C-photosynthate delivered to the enclosed embryo wasestimated to be transferred via the non-vascular region of theseed coat. The cotyledons consist of a single-layered epidermisenclosing storage parenchyma in which a differentiating reticulatevascular system is embedded. Epidermal cells juxtaposed to theseed coat develop wall ingrowths characteristic of transfercells. Initial distribution of ¹⁴C-photosynthate within thecotyledons reflected the unequal delivery to the seed apoplastfrom the vascular and non-vascular regions of the seed coat.Subsequent even distribution of photosynthate within the cotyledonspossibly occurred by transfer within their vascular system. Key words: Cellular pathway, photosynthate transfer, seed anatomy, transfer cell     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> outer ovule integument morphogenesis: Ectopic expression of <Emphasis Type="Italic">KNAT1</Emphasis>reveals a compensation mechanism

Background  

The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated.     

Sink to source translocation in soybean

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [¹⁴C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [¹⁴C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Diurnal water balance of the cowpea fruit

The vascular network of the cowpea (Vigna unguiculata [L.] Walp.) fruit exhibits the anatomical potential for reversible xylem flow between seeds, pod, and parent plant. Feeding of cut shoots with the apoplast marker acid fuchsin showed that fruits imported regularly via xylem at night, less frequently in early morning, and only rarely in the afternoon. The dye never entered seeds or inner dorsal pod strands connecting directly to seeds. Root feeding (early morning) of intact plants with ³²PO₄ or ³H₂O rapidly (20 min) labeled pod walls but not seeds, consistent with uptake through xylem. Weak subsequent (4 hours) labeling of seeds suggested slow secondary exchange of label with the phloem stream to the fruit. Vein flap feeding of subtending leaves with [¹⁴C]sucrose, ³H₂O, and ³²PO₄ labeled pod and seed intensely, indicating mass flow in phloem to the fruit. Over 90% of the ¹⁴C and ³H of fruit cryopuncture phloem sap was as sucrose and water, respectively. Specific ³H activities of transpired water collected from fruits and peduncles were assayed over 4 days after feeding ³H₂O to roots, via leaf flaps, or directly to fruits. The data indicated that fruits transpired relatively less xylem-derived (apoplastic) water than did peduncles, that fruit and peduncle relied more heavily on phloem-derived (symplastic) water for transpiration in the day than at night, and that water diffusing back from the fruit was utilized in peduncle transpiration, especially during the day. The data collectively support the hypothesis of a diurnally reversing xylem flow between developing fruit and plant.     

Seed growth rate and carbohydrate pool sizes of the soybean fruit

The relationships between various carbohydrate pools of the soybean (Glycine max [L.] Merrill) fruit and growth rate of seeds were evaluated. Plants during midpod-fill were subjected to various CO₂ concentrations or light intensities for 7 days to generate different rates of seed growth. Dry matter accumulation rates of seeds and pod wall, along with glucose, sucrose, and starch concentrations in the pod wall, seed coat, and embryo were measured in three-seeded fruits located from nodes six through ten. Seed growth rates ranged from 4 to 37 milligrams·day⁻¹·fruit⁻¹. When seed growth rates were greater than 12 milligrams·day⁻¹·fruit⁻¹, sucrose concentration remained relatively constant in the pod wall (1.5 milligrams·100 milligrams dry weight⁻¹), seed coat (8.5 milligrams·100 milligrams dry weight⁻¹), and embryo (5.0 milligrams·100 milligrams dry weight⁻¹). However, sucrose concentrations decreased in all three parts of the fruit as growth rate of the seeds fell below 12 milligrams·day⁻¹·fruit⁻¹. This relationship suggests that at high seed growth rates, flux of sucrose through the sucrose pools of the fruit was more important than pool size for growth. Starch concentration in the pod wall remained relatively constant (2 milligrams·100 milligrams dry weight⁻¹) at higher rates of seed growth but decreased as seed growth rates fell below 12 milligrams·day⁻¹·fruit⁻¹. This suggests that pod wall starch may buffer seed growth under conditions of limiting assimilate availability. There was no indication that carbohydrate pools of the fruit were a limitation to transport or growth processes of the soybean fruit.