Embryonic Cotyledons Research Articles

Auxin transport into cotyledons and cotyledon growth depend similarly on the ABCB19 Multidrug Resistance‐like transporter

ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resistance-like (MDR) or B group of the ATP-binding cassette (ABC) transporter superfamily, and mediates polar auxin transport in stems and roots. Here we have investigated the role of ABCB19 and auxin distribution in cotyledon development. During embryogenesis, confocal microscopy showed ABCB19 protein to be present in cotyledons during their main growth phase, but not later. Analysis of ProDR5:GFP expression patterns showed a significantly diminished and restricted auxin distribution pattern in abcb19 cotyledons. Nonetheless, development of abcb19 embryonic cotyledons was very similar to that of wild-type. Post-germination, ABCB19 was present in the plasma membrane of cotyledon epidermal, mesophyll and petiole cells during blade expansion. Post-germination cotyledon blade expansion in abcb19 was 65% slower than in wild-type, although the epidermal cell area was reduced by only 17%. The growth rate reduction quantitatively correlated with reduced auxin levels rather than auxin sensitivity as indicated by quantitative ProDR5:GUS assays and direct auxin measurements, and may be explained by the 50% reduction in the import of auxin through the petioles of abcb19 cotyledons during the period of maximum expansion. Taken together, these data indicate that cotyledon expansion during the establishment of photoautotrophic growth depends on ABCB19-mediated auxin import.

DORNRÖSCHENis a direct target of the auxin response factor MONOPTEROS in theArabidopsisembryo

DORNROSCHEN (DRN), which encodes a member of the AP2-type transcription factor family, contributes to auxin transport and perception in the Arabidopsis embryo. Live imaging performed with transcriptional or translational GFP fusions shows DRN to be activated in the apical cell after the first zygotic division, to act cell-autonomously and to be expressed in single cells extending laterally from the apical shoot stem-cell zone at the position of incipient leaf primordia. Here, we show that the Auxin response factor (ARF) MONOPTEROS (MP) directly controls DRN transcription in the tips of the embryonic cotyledons, which depends on the presence of canonical Auxin response elements (AuxREs), potential ARF-binding sites flanking the DRN transcription unit. Chromatin immunoprecipitation experiments show that MP binds in vivo to two AuxRE-spanning fragments in the DRN promoter, and that MP is required for expression of DRN in cotyledon tips. Hence, DRN represents a direct target of MP and functions downstream of MP in cotyledon development.

The content of abscisic acid and the activities of proteinases and trypsin inhibitory proteins, in the germinating seed of common beans under water stress conditions

Specific features of changes in the contents of free and bound abscisic acid (ABA) and the activities of neutral and alkaline proteinases and trypsin inhibitory proteins were determined in the embryonic axis and cotyledons of the common bean (Phaseolus vulgaris L.) after drying. The changes in ABA content, observed following the loss of 5% seed weight, were regarded as an adaptive reaction to stress, whereas the corresponding changes after the loss of 10% of the seed’s weight, was regarded as a result of a pathological disturbance of ABA metabolism. Both drying modes had a negative effect on the state of the proteinase-inhibitory system, as was apparent from the disruption of the regular inverse correlation between the activities of proteinases and serine proteinase inhibitory proteins. A comparison of the dynamics of these characteristics with the buildup of water stress demonstrates an inverse correlation between the content of free ABA and the activity of the proteinases studied. This suggests a potential inhibitory effect of this hormone on the function of the hydrolases in question in the germinating seed.

Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction

AtOPT promoter-GUS fusions were constructed for six of the nine known, putative oligopeptide transporters (OPTs) in Arabidopsis thaliana and used to examine AtOPT expression at various stages of plant development. AtOPT1, AtOPT3, AtOPT4, AtOPT6 and AtOPT7 were expressed in the embryonic cotyledons prior to root radicle emergence. Except for AtOPT8, which gave weak expression, all AtOPTs were strongly expressed in post-germinative seedlings with strongest expression in vascular tissues of cotyledons and hypocotyls. Preferential expression of AtOPTs in vascular tissues was also observed in cotyledons, leaves, hypocotyls, roots, flowers, siliques, and seed funiculi of seedlings and adult plants. Differential tissue-specific expression was observed for specific AtOPTs. For example, AtOPT1, AtOPT3 and AtOPT8 were uniquely expressed in pollen. Only AtOPT1 was expressed in growing pollen tubes, while only AtOPT6 was observed in ovules. AtOPT8 was transiently expressed in seeds during early stages of embryogenesis. Iron limitation was found to enhance expression of AtOPT3. These data suggest distinct cellular roles for specific AtOPTs including nitrogen mobilization during germination and senescence, pollen tube growth, pollen and ovule development, seed formation and metal transport.

Germination and Embryo Anatomy of Osmotically Primed Parsley Schizocarps

`Moss Curled' parsley [Petroselinum crispum (Mill.) Nyman ex. A.W. Hill] schizocarps were osmotically primed in polyethylene glycol at -1.0 MPa for 7 days at 20 °C. The smaller of the two mericarps within a parsley schizocarp had lower germination percentage, but similar rate and synchrony of germination. Osmotic priming increased germination percentage, rate, and synchrony, irrespective of mericarp half. This promotive effect of priming on germination was associated with embryonic advancement as indicated by a doubling of radicle and cotyledon volumes, without changes in lengths of these organs. Periclinal divisions of the lateral expansion meristem, distinct in primed radicles but indistinct in nonprimed radicles, led to radial alignment of the cortical cells and a doubling of cortical volume and thereby increased radicle volume. Each embryonic cotyledon of primed mericarps had three distinct procambial bundles that differentiated along most of the cotyledon length, while nonprimed cotyledons had from zero to three that differentiated only a short way into the cotyledon. Priming increased coyledonary procambium length by 5-fold and volume by 11-fold. Increased embryonic growth due to priming was associated with greater endosperm depletion adjacent to the embryo. The schizocarps frequently separated or partially separated into component mericarps during priming, indicating a weakening of pericarp tissue along the commissural suture and possibly elsewhere.

Anatomical and morphological studies of seed development in Sandersonia aurantiaca (Hook.)

Seed development, including the development of the embryo, the endosperm and the seed coat of Sandersonia aurantiaca (Hook.) has been studied. Seed development in S. aurantiaca takes about 60 days from pollination to maturity. The embryo passes through the early globular, the late globular and the elongated spheroidal stages before reaching the linear embryo stage. Endosperm development conforms to the nuclear type. The cellularisation of the endosperm starts at about 14 days after pollination (DAP) when the embryo reaches the early globular stage, and terminates at about 21 DAP when the embryo is in the late globular stage. The mature seed is globose with a strophiole around the micropyle. The embryo is linear and shows a prominent radicle, a small embryonic axis, shoot apex and large cotyledon. The endosperm constitutes most of the seed volume and contains cells with thickened walls and many storage bodies. The seed coat of a mature seed differentiates into three layers: the epidermis, the subepidermal parenchyma (middle layer), and the lignified layer. Both epidermis and subepidermis come from the outer integument. The lignified layer is derived from both the outer and inner, or only the inner integument.

High Efficiency Organogenesis in Sweet Pepper (Capsicum annuum L.) Tissues from Different Seedling Explants

In vitro plant regeneration was achieved from eightsweet pepper varieties (Capsicum annuum L.). The effect ofvarious explant types (cotyledons, leaves, cotyledonary nodes and shoot-tip from25-day-old seedlings and embryonic cotyledons, embryonic hypocotyls and woundedseedlings) on bud and shoot regeneration and shoot elongation was evaluated.Differences in ability for in vitro shoot regeneration andelongation depended on the variety and explant type. In general, highregeneration frequency was obtained from all varieties. “Agridulce”displayed the highest regeneration response: an average of 3.45 elongated shootsper explant using embryonic cotyledons. Elongated shoots were excised and rootedon Murashige and Skoog (MS) basal medium either without plant growth regulatorsor with 0.5 IBA (indole-3-butyric acid) or 0.05 NAA (α-naphthaleneacetic acid). Plantlets weretransplanted to soil and acclimatised in the greenhouse showing normaldevelopment and growing to maturity bearing normal fruits with seeds.

Tissue-specific localization of mRNA for carrot homeobox genes, CHBs, in carrot somatic embryos.

We examined spatial and temporal expression patterns of four carrot HD-Zip I homeobox genes in somatic embryos. The mRNAs for CHB3, CHB4 and CHB5 were accumulated preferentially in the innermost cortical cell layers of the embryo axis in the torpedo-shaped embryo. In contrast, the accumulation of CHB6 mRNA was restricted to procambial cells of the heart- and torpedo-shaped embryos. In the embryonic cotyledons and the hypocotyl of the seedlings, all of the mRNAs for the four genes were located in the vascular tissues. These findings indicate that different HD-Zip I homeobox genes may be involved in the differentiation of specific tissues during somatic embryogenesis.

Changes in protein composition in cellular membranes of various parts of secondary dormant cucumber seeds treated with ethanol

AbstractSecondary seed dormancy in cucumber (Cucumis sativus L.) cv.Poinsett 76 could be broken with ethanol. Breakage of dormancy was accompanied by significant changes in the composition of membrane proteins in various seed parts. Compared to the intracellular membranes, a change in the protein composition of plasma membranes from the embryonic axis and cotyledons was evident early in the time-course of ethanol-induced germination of dormant seeds. While few alterations in the membrane protein composition in response to ethanol treatment occurred both in dormant and nondormant seeds, a number of changes occurred exclusively in ethanol-treated dormant seeds. Notably, a 14 kD protein in the plasma membrane from the embryonic axis and a 35 kD protein in the microsomal membranes from the perisperm-endosperm envelope disappeared in the ethanol-treated dormant seeds, but not in the ethanol-treated nondormant seeds. Also marked decrease in the content of a 23 kD protein in the plasma membrane from the cotyledons was observed in ethanol-treated dormant seeds only. It is suggested that the particular changes in cellular membrane proteins, which occurred in ethanol-treated dormant seeds much before the first visible indication of germination, might be related to dormancy breaking rather than to the germination process.

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis.

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

Cell cycle events in developing neem (Azadirachta indica) seeds: are they related to intermediate storage behaviour?

AbstractSeeds of neem (Azadirachta indicaA. Juss.), a multipurpose tropical tree, have limited desiccation tolerance. Whether their intermediate storage behaviour might be caused by an elevated percentage of 4C nuclei in the embryo at maturity was investigated. Morphological development of neem seeds was monitored on selected trees in Ouagadougou, Burkina Faso. Developing seeds were tested for germinability, and cell cycle events were determined by using flow cytometry and analysing the level of β-tubulin. Germination could occur after 8 weeks of seed development, but normal seedlings resulted only after 10 weeks or more. The change in fruit colour from green to yellow began after approximately 12 weeks of seed development. Immature, 4-week-old embryos about 2 mm in length had 15% of cells in the G2phase and 60% in the G1phase of the cell cycle, as indicated by 4C and 2C nuclear DNA levels, respectively. During maturation, the proportion of G2cells declined to 3% at full maturity and slightly further after drying, and the proportion of G1cells increased to approximately 90%. A strong β-tubulin signal was observed in tips of young embryonic radicles and cotyledons, but a weak or non-detectable signal was found in 9-week-old ones and in those from green-mature and yellow fruits. Because DNA replication and β-tubulin level were almost negligible at seed maturity, as in orthodox tomato seeds, it is suggested that these factors are not involved in the intermediate storage behaviour of neem seeds.

A comparison of desiccation-related proteins (dehydrin and QP47) in peas (Pisum sativum)

AbstractDehydrin and QP47, proteins present in mature pea seeds (Pisum sativum), have been proposed to play protective roles during desiccation. To identify possible relationships between these proteins and desiccation tolerance, their tissue locations and patterns of synthesis and degradation have been examined during germination. Tissue locations were determined by immunocytochemistry using polyclonal antibodies raised against a conserved dehydrin amino acid sequence and against purified QP47. In embryonic axis and cotyledon cells, QP47 and dehydrin were distributed uniformly with no apparent nuclear or organellar specificity. Both proteins were present in 24 h-imbibed axes that had not initiated radicle growth but were completely absent from 24 h-imbibed axes that had begun to grow. The amounts of QP47 and dehydrin in embryonic axes decreased with time after the start of imbibition and were undetectable by 48 h. When germination was prevented by polyethylene glycol (PEG) or abscisic acid (ABA), both proteins remained at their original amounts. Thus, both QP47 and dehydrin disappeared coincidently with the beginning of growth and not simply as a function of the time after imbibition. QP47 persisted in cotyledons until at least 31 days into seedling growth, whereas dehydrin was not detectable in cotyledons after 7 days. Dehydrin, but not QP47, could be re-induced in pea shoots and cotyledons by dehydration. The timing of degradation of both proteins was correlated with the loss of desiccation tolerance during germination of pea axes.

Auxin-regulated changes of fatty acid content and composition in soybean zygotic embryo cotyledons

The present studies were conducted to investigate the effect of exogenous auxins on zygotic embryo fatty acid content and lipid composition during somatic embryo induction. Immature soybean ( Glycine max (L.) Merr.) cotyledons from two developmental stages were cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The presence of 2,4-D or NAA in the medium altered the content and percentage of oleic (18:1), linoleic (18:2) and linolenic (18:3) acids, with less effects on stearic (18:0), palmitic (16:0) or total fatty acids in the embryonic cotyledons. Desaturation assays with radiolabelled fatty acids showed that 2,4-D increased both the ratios of 18:2 to 18:1 and 18:3 to 18:2. NAA at 10 mg/l resulted in an increase in the content and percentage of 18:3 but not that of 18:2 in the embryonic cotyledons. Analysis of free fatty acids, triglycerides, phosphatidylcholine, phosphatidylethanolamine, and glycolipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol) revealed that the increased ratio of 18:3 to 18:2 occurred in phosphatidylcholine, triglycerides and free fatty acids, but not in glycolipids. Our data suggest that 2,4-D increased the efficiency of the desaturation related processes in extra plastidial compartments.

Phytate Metabolism in Bean Seedlings duringPost-Germinative Growth

Summary The phytate metabolism of snap and pinto beans seedlings was studied during post-germinativegrowth. Rapid phytate degradation in embryonic axis started at the time of root emergence and continued for two days of post-germinative growth. Surprisingly, phytate synthesis was observed in the embryonic axes of both races after 2 – 3 days and the original amount of phytate for the embryonic axis had doubled the amount in the axis of dry seed. The phytate synthesis was inferred because the phytate level in embryonic axis reached a plateau per unit of fresh weight after 3 days of post-germinative growth. Inorganic phosphate (P i ) at the 10 and 20 mM level significantly increased the endogenous P i content in the embryonic axes, but showed no effect on the phytate levels in the embryonic axes or cotyledons. The phytate content of bean embryonic axes underwent a significant increase upon cultivation with extremely high levels of P i (40 mM). A similar effect was observed using lower P i levels (20 mM) if the axes had been excised prior to cultivation. Both treatments resulted in the phytate accumulation, but adverse effects on the growth and development of embryonic axes were associated with both these treatments. We conclude that phytate synthesis in the embryonic axis is a regular event during post-germinative growth of bean seedlings. Furthermore, the phytate degradation in cotyledons reflected the growth rate of the embryonic axis irrespectively of the Pi content in the embryonic axis.

Plantlet regeneration from mature zygotic embryos and embryonic explants of masson pine (Pinus massoniana Lamb.)

Excised zygotic embryos, cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones. BA (1.0 mg/ L) in combination with NAA (0.05 mg/L)in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos, but most of them were formed at the tips of embryonic cotyledons. Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L. Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal (0.5%). Root initiation was achieved with full or half strength DCR medium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L. Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated (3-20 h) with BA 50-100 mg/L, followed by transfer to hormone-free DCR medium. The maximum number of shoots obtained per explant within six months was 33.

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.

The effect of ethylene on RNA and protein synthesis in germinating Manketti (Ricinodendron rautanenii Schinz) seeds

Amino acid uptake and protein synthesis were monitored in the embryonic axis, cotyledons and endosperm of manketti seeds (Ricinodendron rautanenii Schinz) during imbition, dormancy and dormancy-breaking by ethylene. Protein synthesis increased in all tissues within 48 h of imbibition and was strongly inhibited by cycloheximide and actinomycin D. No marked increase in protein synthesis was observed following ethylene treatment suggesting that qualitative rather than quantitative changes in protein synthesis were associated with dormancy-breaking. Seed germination was relatively insensitive to treatment with actinomycin D in marked contrast to the inhibition observed with cycloheximide.

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

Polyamine Titer in the Embryonic Axis and Cotyledons of <i>Glycine max</i> (L.) during Seed Growth and Maturation

Caffeine hazards and their prevention in germinating seeds of coffee (Coffea arabica L.)

The inhibition of growth of seedlings of coffee (Coffea arabica L.) exposed to 10 m M caffeine was found to occur in the rootlet: mitosis and cell plate formation were also inhibited. Since concentrations of endogenous caffeine in the imbibed seed are 40-60 mM, 4-6 times as high as in the seedlings, we conclude that coffee embryos have specific means of avoiding caffeine autotoxicity. Observations indicate that cell divisions in root tips start only after the latter are pushed away from the caffeine-rich endosperm by elongation of the hypocotyl and maintained through cell elongation. Caffeine is introduced into the embryonic cotyledons mostly after cell division is completed there. Thus, coffee seedlings may avoid autotoxic effects of endogenous caffeine by separation between sites where mitosis is occurring and those where caffeine is stored. This is achieved in root tips by separation is space but in the cotyledons by separation in time. Caffeine is liberated from the tree litter in coffee plantations and eventually will produce autotoxic effects, resulting in some degeneration.

Respiration during seed maturation

The contribution of cytochrome and alternative pathways to respiration during soybean [Glycine max (L.) Merr. cv. Chippewa 64] seed formation and maturation was measured by oxygen consumption of embryonic axis and cotyledon tissues and of mitochondria isolated from immature seeds. Respiration rates (dry weight basis) declined duing the last month of seed maturation. Cyanide sensitive respiration (cytochrome pathway) accounted for 80 to 90% of total oxygen consumption throughout seed formation and maturation. The proportion of the total respiration resistant to 1 mM cyanide and inhibited by 1 mM salicylhydroxamic acid (alternative pathway) did not exceed 6%. A residual oxygen consumption, accounting for 5 to 15% of total respiration, persisted in the presence of both inhibitors. Likewise, virtually no alternative respiration was found in mitochondria prepared from immature seeds. The alternative respiratory pathway seems absent during soybean seed formation and maturation regardless of whether grown in the greenhouse or field.