Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Ontogenetic variation of four cytokinins in soybean root pressure exudate

Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.

Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.

No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).

The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean.

     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Cytokinin Biochemistry in Relation to Leaf Senescence: IV. Cytokinin Metabolism in Soybean Explants

When [³H]dihydrozeatin riboside and [³H]zeatin riboside were supplied to soybean (Glycine max L.) explants (comprising one leaf, associated pods, and subtending stem) via the xylem at mid to late podfill, 0.1% of the supplied ³H was extracted from the seeds. The distribution of ³H in the explants was similar to that bound previously following uptake of [³H]zeatin riboside at earlier stages of pod development. Metabolites formed in the explants from ³H-labeled zeatin, zeatin riboside, and dihydrozeatin riboside were identified and related to the endogenous cytokinins shown to be present. When zeatin riboside and zeatin were supplied for 1 hour, zeatin nucleotide was the principal metabolite formed and this appeared to be the precursor of the other metabolites detected subsequently. Explants supplied with zeatin riboside or dihydrozeatin riboside for 1 hour, and then transferred to water for 20 to 24 hours, yielded leaf blades in which the main metabolites were O-glucosyldihydrozeatin, adenosine, and adenine. The metabolism of zeatin riboside in blades of explants at pre-podfill, early podfill, and mid to late podfill did not differ appreciably. The results are discussed in relation to leaf senescence and seed development.     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Identification of Cytokinins from Xylem Exudate of Phaseolus vulgaris L

The principal biologically active cytokinins in xylem exudate of young Phaseolus vulgaris L. plants were identified by bioassay, high-performance liquid chromatography, enzymic degradation and combined gas chromatography-mass spectrometry (selected ion monitoring) a zeatin riboside, zeatin nucleotide, dihydrozeatin riboside, dihydrozeatin nucleotide, O-glucosyl zeatin, O-glycosyl dihydrozeatin, O-glucosyl dihydrozeatin riboside, and O-glucosyl dihydrozeatin nucleotide. Trace amounts of O-glucosyl zeatin riboside and O-glucosyl zeatin nucleotide were also detected.     

Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean

Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

The Physiological Basis for Cytokinin Induced Increases in Pod Set in IX93-100 Soybeans

Previous investigations have shown the feasibility of increasing pod number on legumes by the application of 6-benzylaminopurine (BA) directly to the raceme. These investigations were designed to determine what reproductive parameter was affected by cytokinin application, and if these applications were overcoming a deficiency in root-produced cytokinins during late flowering. Five individual main stem racemes on greenhouse grown soybeans (Glycine max L. Merr.) were treated with 2 millimolar BA. A single application of BA when pods appeared at 25 to 50% of the proximal floral positions resulted in a 58% increase in pod set due primarily to a 33% reduction in floral abscission. Applications of BA at later intervals also resulted in significant reductions in total abscission. When three applications of BA were imposed on the upper five nodes of field grown soybeans, total pod number and seed weight were significantly increased in this section of the canopy by 27 and 18%, respectively. Throughout the flowering period, root pressure exudate was sampled for the subsequent separation and quantification of zeatin, dihydrozeatin, zeatin riboside, dihydrozeatin riboside, and isopentenyladenine. Total cytokinin flux peaked from 0 to 9 days after flowering began, and then dropped to one-half of this level by 15 days postanthesis. The probability that a flower would initiate a pod was directly related to the concentration of total cytokinins present in the exudate when the flower opened.     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

Radioimmunoassay for quantifying the cytokininscis-zeatin andcis-zeatin riboside and its application to xylem sap samples

An antiserum against the cytokinincis-zeatin riboside was raised in rabbits and characterized for use in radioimmunoassays. Cross-reactivity studies demonstrated the specificity of the selected antiserum forcis-zeatin riboside andcis-zeatin in preference to a range of cytokinins and other purines. HPLC systems were developed that separatedcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively. These systems enabled the separation of these compounds in xylem sap samples of wheat and oats and their quantification using radioimmunoassay. A TLC system for the separation ofcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively, is also described.     

Effect of cytokinins supplied via the xylem at multiples of endogenous concentrations on transpiration and senescence in derooted seedlings of oat and wheat

This study was conducted lo determine whether naturally occurring xylem cytokinins, when supplied to leaves via the xylem at approximately endogenous concentrations, increase transpiration and delay senescence in selected monocot species (oat and wheat). The concentrations of some of the major cytokinins (zeatin, dihydrozeatin, ciszeatin and their ribosides, the O-glucosides and nucleotides) were determined in the xylem exudate of oat and wheat seedlings by radioimmunoassay. Evidence is presented that the small volume of exudate (4–5 mm³) collected per plant was xylem sap in transit at the time of shoot excision. Using the data on cytokinin levels, the individual bases and ribosides (and a base/riboside mixture), at multiples of concentrations determined in xylem sap, were tested in transpiration and senescence bioassays. The individual O-glucosides (and mixtures of the O-glucosides) were similarly tested at (i) multiples of the molar concentrations of the corresponding bases and ribosides, and/or at (ii) multiples of the endogenous concentrations. Similarly, zeatin and dihydrozeatin nucleotides were tested at multiples of the molar concentration of zeatin riboside and, in some instances, at multiples of endogenous concentrations. Our results suggest that, at least in oat and possibly in wheat, zeatin-type bases, ribosides and O-glucosides supplied to the leaf in xylem sap are likely to play a role in regulating transpiration in vivo. O-glucosides in oat xylem sap may be important regulators of leaf senescence in the intact plant. The nucleotides were present in xylem sap at lower concentrations than most of the bases, ribosides and O-glucosides. The nucleotides appear likely to play a lesser role than the bases, riboside and O-glucosidcs in controlling transpiration and senescence in the intact plant.     

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida , their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC-MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin-type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)-type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin-like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP-compounds, suggesting either import of iP-type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.     

Cytokinins in maturing and germinatingLupinus luteus L. seeds

³H-labelled zeatin riboside (ZR) was applied to pod walls of intactLupinus luteus L. plants. Metabolites present in mature, dry seeds were zeatin nucleotide (ZNT), zeatin riboside (ZR) and zeatin (Z), zeatin O-glucosides and lupinic acid (LA), and the corresponding dihydro-derivatives of the cytokinins listed. Endogenous cytokinins were rapidly metabolised in germinating seeds. In seeds labelled with [³H]ZR for 90 min following a 2 h period of imbibition in water, ZR was actively converted to ZNT and dihydro-ZNT but the prevailing CTK was Z in cotyledons and ZR in embryo axes (EA); later LA and dihydro-LA, and O-glucoside metabolites accumulated. When [³H] zeatin was introduced into imbibing seeds, it was converted to dihydro-ZNT, ZNT, dihydro-ZR, ZR and dihydro-Z; in EA of the Z-labelled seeds, dihydro-ZR and ZR were the main cytokinins. After incubation of the Z-labelled seeds for 6 h in water, the ratios of dihydro-ZNT: ZNT and dihydro-ZR: ZR were, respectively, 20: 1 and 3.4: 1 in EA, and 3.5: 1 and 1.4: 1 in cotyledons.