Linking ion transport, electron flow and oxidative phosphorylation; one aspect of the research of Professor Sir Rutherford Ness Robertson, AC, KCMG, DSc, FAA, FRS (1913-2001).

A summary is presented of the work on ion transport of Professor Sir Rutherford Ness Robertson.

The sites of interaction of triphenyltetrazolium chloride with mitochondrial respiratory chains.

The inability of cells and microorganisms to reduce the colourless electron acceptor triphenyltetrazolium chloride (TTC) to a red formazan precipitate is commonly used as a means of screening for cells that have a dysfunctional respiratory chain. The site of reduction of TTC is often stated to be at the level of cytochrome c oxidase where it is assumed to compete with oxygen for reducing equivalents. However, we show here that TTC is reduced not by cytochrome c oxidase but instead by dehydrogenases, particularly complex I, probably by accepting electrons directly from low potential cofactors. The reduction rate is fastest in coupled membranes because of accumulation in the matrix of the positively charged TTC+ cation. However, the initial product of TTC reduction is rapidly reoxidised by molecular oxygen, so that generation of the stable red formazan product from this intermediate occurs only under strictly anaerobic conditions. Colonies of mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions to allow the intermediate to form the stable red formazan. This revision of the mode of interaction of TTC with respiratory chains has implications for the types of respiratory-defective mutants that might be detected by TTC screening.

Regulation of respiration in rotenone-treated tobacco cell suspension cultures.

Cells of Nicotiana tabacum L. suspension cultures were treated with the respiratory inhibitor rotenone, which specifically inhibits complex I activity of mitochondria. Rotenone retarded cell growth, as shown by decreases in fresh weight, dry weight and cell numbers on a suspension-volume basis. However, rates of the coupled respiration were higher in rotenone-treated compared to control cells when expressed on a fresh-weight basis. Rates of the rotenone-insensitive respiration increased substantially on both a fresh-weight and extractable-cellular-protein basis 24 h after rotenone treatment. ATP/ADP ratios were not significantly different between control and rotenone-treated cells. Our results indicated that cells of tobacco suspension cultures were able to maintain a slow rate of growth and adequate ATP/ADP ratios without the operation of complex I.

The production of an inducible antisense alternative oxidase (Aox1a) plant.

Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system.

A strain of Synechocystis sp. PCC 6803 without photosynthetic oxygen evolution and respiratory oxygen consumption: implications for the study of cyclic photosynthetic electron transport.

Cyclic electron transport around photosystem (PS) I is believed to play a role in generation of ATP required for adaptation to stress in cyanobacteria and plants. However, elucidation of the pathway(s) of cyclic electron flow is difficult because of low rates of this electron flow relative to those of linear photosynthetic and respiratory electron transport. We have constructed a strain of Synechocystis sp. PCC 6803 that lacks both PSII and respiratory oxidases and that, consequently, neither evolves nor consumes oxygen. However, this strain is still capable of cyclic electron flow around PSI. The photoheterotrophic growth rate of this strain increased with light intensity up to an intensity of about 25 mumol photons m-2 s-1, supporting the notion that cyclic electron flow contributes to ATP generation in this strain. Indeed, the ATP-generating ability of PSI is demonstrated by the fact that the PSII-less oxidase-less strain is able to grow at much higher salt concentrations than a strain lacking PSI. A quinone electrode was used to measure the redox state of the plastoquinone pool in vivo in the various strains used in this study. In contrast to what is observed in chloroplasts, the plastoquinone pool was rather reduced in darkness and was oxidized in the light. This is in line with significant electron donation by respiratory pathways (NADPH dehydrogenase and particularly succinate dehydrogenase) in darkness. In the light, the pool becomes oxidized due to the presence of much more PSI than PSII. In the oxidase-less strains, the plastoquinone pool was very much reduced in darkness and was oxidized in the light by PSI. Photosystem II activity did not greatly alter the redox state of the plastoquinone pool. The results suggest that cyclic electron flow around PSI can contribute to generation of ATP, and a strain deficient in linear electron transport pathways provides an excellent model for further investigations of cyclic electron flow.

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.

The nuclear origin of the non-phosphorylating NADH dehydrogenases of plant mitochondria.

The oxidation of matrix and cytosolic NADH by isolated beetroot and wheat leaf mitochondria was investigated to determine whether the rotenone-insensitive NADH dehydrogenases of plant mitochondria were the products of nuclear or mitochondrial genes. After aging beetroot tissue (slicing and incubating in a CaSO4 solution), the induction of the level of matrix NADH oxidation in the presence of rotenone was greatly reduced in mitochondria isolated from tissue treated with cycloheximide, a nuclear protein synthesis inhibitor. This was also true for the oxidation of cytosolic NADH. Mitochondria isolated from chloramphenicol-treated tissue exhibited greatly increased levels of both matrix and external rotenone-insensitive NADH oxidation when compared to the increase due to the aging process alone. This increase was not accompanied by an increase in matrix NAD-linked substrate dehydrogenases such as malic enzyme nor intra-mitochondrial NAD levels. Possible explanations for this increase in rotenone-insensitive NADH oxidation are discussed. Based on these results we have concluded that the matrix facing rotenone-insensitive NADH dehydrogenase of plant mitochondria is encoded by a nuclear gene and synthesis of the protein occurs in the cytosol.

Activation of the plant alternative oxidase by high reduction levels of the Q-pool and pyruvate.

This report describes the activation of the alternative oxidase (AOX) of higher plant mitochondria by a high reduction level of the ubiquinone pool in the presence of pyruvate. In mitochondria from both thermogenic (Arum italicum spadices) and nonthermogenic (Glycine max cotyledons) tissues AOXis activated when the Q-pool becomes highly reduced in the presence of pyruvate. Pyruvate is essential for this activation. The enzyme is not activated when pyruvate is added after a transient high reduction level of the Q-pool, but is when pyruvate is added before the transient reduction. Pyruvate also protects the enzyme against inhibition during catalytic turnover. Although this activation is not accompanied by a reduction of the covalent disulfide bond, the same activation can be achieved with dithiothreitol (DTT). It is suggested that a part of the activation by DTT is not the result of reducing the covalent disulfide bond, and the relation between these types of activation is discussed. The importance of this activation for the in vivo regulation and its relation to previously reported activators is discussed. A mechanism is proposed in which it is suggested that AOX is inactivated by its product (oxidized ubiquinone) during catalysis and that this inhibition is prevented in the presence of pyruvate. The inhibition can be reversed by a reductive process, achieved by high levels of reduction of the Q-pool or by DTT, but not by pyruvate. This restoration of activity is not related to the redox process involved in reducing the covalent disulfide bond.

Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase.

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.

An NADP-glutamate dehydrogenase from the green alga Bryopsis maxima. Purification and properties.

NADP-glutamate dehydrogenase (EC 1.4.1.4:NADP-GDH) was purified to electrophoretic homogeneity from the multinuclear-unicellular green marine alga in Siphomales, Bryopsis maxima, and its properties were examined. M(r) of the undenatured enzyme was 280 kDa, and the enzyme is thought to be a hexamer of 46 kDa subunit protein. Optimum pHs for the reductive amination and oxidative deamination were 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specific activities with a ratio of 18:1. Apparent K(m) values for 2-oxoglutarate, ammonia, NADPH, glutamate and NADP+ were 3.0, 2.2, 0.03, 3.2 and 0.01 mM respectively. The enzymochemical characteristics of the GDH were studied and compared to those of other species. The B. maxima GDH was insensitive to 5 mM Ca(2+) and to 1 mM EDTA in contrast to higher plant NAD-GDHs. Chemical modifications with DTNB and pCMBS suggested that cysteine residues are essential for the enzymatic activity as in other species GDHs. The GDH was not affected by 1 mM purine nucleotides, suggesting that the enzyme is not allosteric, in contrast to animal NAD(P)-GDHs and fungal NAD-GDHs.

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate.

The kinetics of alternative oxidase (AOX) of Arum italicum spadices and soybean (Glycine max L.) cotyledons were studied both with intact mitochondria and with a solubilized, partially purified enzyme. Ubiquinone analogs were screened for their suitability as substrates and ubiquinol-1 was found to be most suitable. The kinetics of ubiquinol-1 oxidation via AOX in both systems followed Michaelis-Menten kinetics, suggesting that the reaction is limited by a single-step substrate reaction. The kinetics are quite different from those previously described, in which the redox state of ubiquinone-10 was monitored and an increase in substrate was accompanied by a decrease in product. The difference between the systems is discussed. Pyruvate is a potent activator of the enzyme and its presence is essential for maximum activity. The addition of pyruvate to the solubilized enzyme increased the maximum initial velocity from 6.2 [plus or minus] 1.3 to 16.9 [plus or minus] 2.8 [mu]mol O2 mg-1 protein min-1 but had little effect on the Michaelis constant for ubiquinol-1, an analog of ubiquinol, which changed from 116 [plus or minus] 73 to 157 [plus or minus] 68 [mu]M. It is concluded that pyruvate (and presumably other keto acids) increases the activity of AOX but does not increase its affinity for its substrate. In agreement with this is the finding that removal of pyruvate (using lactate dehydrogenase and NADH) leads to an 80 to 90% decrease in the reaction rate, suggesting that pyruvate is important in the mechanism of reaction of AOX. The removal of pyruvate from the enzyme required turnover, suggesting that pyruvate is bound to the enzyme and is released during turnover.

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria.

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation.

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation).

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

New inhibitors of the ubiquinol oxidase of higher plant mitochondria.

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1. A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.

Regulation of alternative oxidase activity in higher plants.

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanide-insensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.

Activation of glycine decarboxylase in pea leaf mitochondria by ATP.

Activity of glycine decarboxylase decreased by 60-70% after the isolated pea leaf mitochondria were aged for 5 h in the absence of glycine and was completely lost after 24 h. The reverse reaction, i.e., production of glycine from serine, ammonium, dihydrolipoate, and bicarbonate, was also inhibited in these aged mitochondria. Glycine decarboxylase could be reactivated by both exogenous and endogenous ATP. The latter was formed during the oxidation of succinate, malate, or oxoglutarate. Glycine decarboxylase consists of four subunits (P-, H-, L-, and T-proteins). The aged mitochondria were able to catalyze the exchange of [14C]-bicarbonate-glycine and the oxidation of dihydrolipoate, indicating the persistence of P-, H-, and L-protein activities. Serine hydroxymethyltransferase catalyzes the formation of serine from methylene tetrahydrofolate and another glycine and molecule at the last reaction of glycine oxidation. The aged mitochondria were able to catalyze the formation of methylene tetrahydrofolate from [14C]serine and its reverse reaction. Therefore, it was concluded that the loss of glycine decarboxylase activity was due to an inhibition of the reaction catalyzed by T-protein, which required ATP for its activation.

Cytochrome and alternative respiratory pathways compete for electrons in the presence of pyruvate in soybean mitochondria.

The partitioning of electrons between the alternative oxidase and the cytochrome pathway of soybean mitochondria has been reassessed in the presence of the alternative oxidase activator pyruvate. In the presence of pyruvate and with succinate as substrate, the alternative oxidase became active at a much lower level of ubiquinone reduction than in the absence of pyruvate. Under state 4 (no ADP present) conditions, activation of the alternative oxidase with pyruvate resulted in an oxidation of b cytochromes, demonstrating switching of electrons away from the cytochrome chain. In the presence of ferricyanide and the cytochrome oxidase inhibitor KCN, cytochrome chain activity could be followed spectrophotometrically and that of the alternative pathway with an oxygen electrode. Under these conditions, the addition of pyruvate diverted electron flow from the cytochrome chain to the alternative pathway; subsequent inhibition of the alternative oxidase increased electron flow via the cytochrome chain. This indicates that electrons can be switched from one pathway to the other when the cytochrome chain is not saturated and this was confirmed by n-propylgallate titrations (p plots) of mitochondria oxidizing succinate. Decreases in ADP/O ratios and phosphorylation rate upon addition of pyruvate indicated that the alternative pathway could also contribute to respiration under state 3 conditions. The results indicate that when the alternative oxidase is activated by pyruvate, it can compete for electrons with the cytochrome chain and does not act as an overflow pathway. The significance of these observations for in vivo respiration is discussed.

Ubiquinone redox behavior in plant mitochondria during electron transport.

The redox poise of the ubiquinone pool during electron transfer in isolated soybean mitochondria has been compared using two different procedures: the rapid organic extraction of ubiquinone followed by quantification of the oxidized and reduced forms using high-pressure liquid chromatography and an electrochemical technique that measures ubiquinone reduction voltametrically. The goal of these studies was to rigorously test the use of the voltametric technique to monitor the redox status of ubiquinone during the course of mitochondrial electron transfer. The linear relationship between the two methods confirms the reliability of the data obtained with the voltametric technique; however, redox inactive pools of ubiquinone were detected with the HPLC technique. We also quantified the absolute amounts of the ubiquinone homologues ubiquinone-9 and ubiquinone-10 in mitochondria isolated from different soybean tissues and compared their behavior during electron transfer in the presence and absence of pyruvate, an allosteric effector of the cyanide-resistant electron transfer pathway. Both homologues belong to a common redox-active pool in the inner mitochondrial membrane. The results indicate that ubiquinone can be a limiting component of electron transfer through the cyanide-resistant pathway, particularly in roots where its concentration is much lower than in cotyledons.