Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 µm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

Characterization of foliar manganese (Mn) in Mn (hyper)accumulators using X-ray absorption spectroscopy.

Plant hyperaccumulation of the essential nutrient manganese (Mn) is a rare phenomenon most evident in the Western Pacific region, and differs from hyperaccumulation of other elements. Mn hyperaccumulators employ a variety of species-dependent spatial distribution patterns in sequestering excess foliar Mn, including primary sequestration in both nonphotosynthetic and photosynthetic tissues. This investigation employed synchrotron X-ray absorption spectroscopy (XAS) in a comparative study of Mn (hyper)accumulators, to elucidate in situ the chemical form(s) of foliar Mn in seven woody species from Australia, New Caledonia and Japan. Foliar Mn was found to predominate as Mn(II) in all samples, with strong evidence of the role of carboxylic acids, such as malate or citrate, as complexing ligands. Overall, the X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine-structure spectroscopy (EXAFS) data appeared weighted against previous observations that oxalate binds excess Mn in Mn-(hyper)accumulating species.

Foliar manganese accumulation by Maytenus founieri (Celastraceae) in its native New Caledonian habitats: populational variation and localization by X-ray microanalysis.

Hyperaccumulation by plants is a rare phenomenon that has potential practical benefits. The majority of manganese (Mn) hyperaccumulators discovered to date occur in New Caledonia, and little is known about their ecophysiology. This study reports on natural populations of one such species, the endemic shrub Maytenus founieri. Mean foliar Mn concentrations of two populations growing on ultramafic substrates with varying soil pHs were obtained. Leaf anatomies were examined by light microscopy, while the spatial distributions of foliar Mn in both populations were examined by qualitative scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS). Plants growing on two different substrates were found to have very different mean dry weight (DW) foliar Mn concentrations. Light microscopy showed that the leaves had very distinct thick dermal structures, consisting of multiple layers of large cells in the hypodermis. In vivo X-ray microprobe analyses revealed that, in both populations, Mn sequestration occurred primarily in these dermal tissues. The finding here that foliar Mn is most highly localized in the nonphotosynthetic tissues of M. founieri contrasts with results from similar studies on other woody species that accumulate high Mn concentrations in their shoots.

Variation in defence strategies in two species of the genus Beilschmiedia under differing soil nutrient and rainfall conditions.

The relationships between various leaf functional traits that are important in plant growth (e.g., specific leaf area) have been investigated in recent studies; however, research in this context on plants that are highly protected by chemical defences, particularly resource-demanding nitrogen-based defence, is lacking. We collected leaves from cyanogenic (N-defended) Beilschmiedia collina B. Hyland and acyanogenic (C-defended) Beilschmiedia tooram (F. M. Bailey) B. Hyland at high- and low-soil nutrient sites in two consecutive years that varied significantly in rainfall. We then measured the relationships between chemical defence and morphological and functional leaf traits under the different environmental conditions. We found that the two species differed significantly in their resource allocation to defence as well as leaf morphology and function. The N defended species had a higher leaf nitrogen concentration, whereas the C-defended species had higher amounts of C-based chemical defences (i.e., total phenolics and condensed tannins). The C-defended species also tended to have higher force to fracture and increased leaf toughness. In B. collina, cyanogenic glycoside concentration was higher with higher rainfall, but not with higher soil nutrients. Total phenolic concentration was higher at the high soil nutrient site in B. tooram, but lower in B. collina; however, with higher rainfall an increase was found in B. tooram, while phenolics decreased in B. collina. Condensed tannin concentration decreased in both species with rainfall and nutrient availability. We conclude that chemical defence is correlated with leaf functional traits and that variation in environmental resources affects this correlation.

Manganese accumulation in the leaf mesophyll of four tree species: a PIXE/EDAX localization study.

Little is known about the spatial distribution of excess manganese (Mn) in the leaves of tolerant plants. Recently, the first such study of a Mn hyperaccumulator showed that the highest localized Mn concentrations occur in the photosynthetic tissue. This is in contrast to reports based on localization of foliar accumulation of other heavy metals. Here, four tree species, Gossia bidwillii, Virotia neurophylla, Macadamia integrifolia and Macadamia tetraphylla, which hyperaccumulate or strongly accumulate Mn, were studied. Cross-sectional foliar Mn localization was carried out in situ using proton-induced X-ray emission/energy dispersive X-ray analysis (PIXE/EDAX). All four species contained photosynthetic tissues with multiple palisade layers. These were shown to be the primary sequestration sites for Mn. Mn was not detected in the epidermal tissues. The findings of this study demonstrate a concurrence of three traits in four tree species, that is, accumulation of excess Mn in the leaves, its primary sequestration in the photosynthetic tissues, and multiple-layer palisade mesophyll.

Modelling the role of Rubisco activase in limiting non-steady-state photosynthesis.

The roles of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase in limiting the approach of photosynthesis to steady-state following a step increase from a low to a saturating value of photon flux density (PFD) are reviewed. This information, along with the effect of Rubisco on steady-state photosynthetic rate and the effect of Rubisco activase on maximum Rubisco activation state, is then used to construct a model to predict the optimum allocation of protein between Rubisco and Rubisco activase for plants exposed to different light environments. The model predicts that the distribution of protein that produces the maximum steady-state rate of photosynthesis does not produce the maximum activation rate for Rubisco or the maximum steady-state activation state. The latter conclusion may explain why Rubisco is rarely found to be fully activated in leaves, even at saturating PFD values. The former suggests that plants exposed to fluctuating PFD should allocate more protein to Rubisco activase than plants exposed to constant PFD. This aspect of the model is explored in more detail for lightflecks of differing duration.

Regulation of Rubisco activation in antisense plants of tobacco containing reduced levels of Rubisco activase.

Following an increase in photon flux density (PFD), ribulose bisphosphate carboxylase/oxygenase (Rubisco) undergoes a slow activation which substantially limits the rate of photosynthesis. This activation process is mediated in part by Rubisco activase. Antisense DNA plants of tobacco were used to quantify the degree to which activase limits Rubisco activation. Reductions in leaf activase content caused proportional reductions in the rate of Rubisco activation following a PFD increase from 110 to 1200 micromol m(-2) sec(-1). This was the case for activase levels up to and slightly beyond normal wild-type activase levels. Activase therefore has a flux control coefficient of unity with respect to the Rubisco activation flux. Such a high control coefficient has rarely been measured for any metabolic system, and this is the highest control coefficient measured for an important photosynthetic flux. In contrast, the rate of Rubisco inactivation in leaves following a drop in PFD of 1200 to 110 micromol m(-2) sec(-1) was unchanged by a 60% reduction in activase levels. Despite the high degree of control that activase exerts over the rate of activation, and thus non-steady-state photosynthesis, it was shown that steady-state photosynthesis was largely unaffected by activase concentration until it was reduced below approximately 15% of the wild-type level. The significance of these results and their implications for published models of Rubisco activation are discussed.

The effects of elevated CO2 atmospheres on the nutritional quality of Eucalyptus foliage and its interaction with soil nutrient and light availability.

Seedlings of Eucalyptus tereticornis (Smith) were grown under two levels of availability each of CO2 (352 and 793 µmol mol-1), soil nutrients (1/24 and 1/4 Hoagland's solution) and light (full and 30% sunlight). Low soil nutrient availability or high light increased the C:N ratio of leaves, leading to lower leaf nitrogen concentrations, higher leaf specific weights and higher levels of both total phenolics and condensed tannins. These results were consistent with other studies of the effect of environmental resource availability on foliage composition. Similar results were observed when the C:N ratio of leaves was increased under elevated CO2. The changes in leaf chemistry induced by the treatments affected the performance of 4th-instar larvae of Chrysophtharta flaveola (Chapuis) fed on the leaves. Increased C:N ratios of leaves reduced digestive efficiencies and pupal body sizes and increased mortality. Below a threshold nitrogen concentration of approximately 1% dry mass, severe reductions in the performance of larvae were recorded. Such changes may have significant consequences for herbivores of Eucalyptus, particularly in view of projected increases in atmospheric CO2.

Dependence of the Extent and Direction of Average Stomatal Response in Zea mays L. and Phaseolus vulgaris L. on the Frequency of Fluctuations in Environmental Stimuli.

Stomatal responses to fluctuating light and CO2 were investigated in Zea mays and Phaseolus vulgaris. Slow-moving stomata can affect carbon gain and water loss by plants during light flecks, under dynamic cloud cover, during alternating windy and calm air conditions (which influence CO2 concentrations and humidity immediately around leaves in plant canopies), at natural CO2 vents, or in growth chambers with imperfect CO2 control. It was found that the frequency of constant-amplitude fluctuations in light and CO2 dramatically affected the time-averaged stomatal conductance in both Zea and Phaseolus. During oscillations in light, average stomatal conductance was driven either above or below that observed at steady state at the average light level, depending on the frequency of the oscillations. Under oscillating CO2, the departure of average stomatal conductance away from that observed at steady state at the average CO2 level was also frequency dependent in both species. Upon cessation of oscillations and return of light or CO2 to the stable median level, stomatal conductance also returned to a steady state, matching that before oscillations were initiated. This work shows that fluctuations in light and CO2, and equally important, their frequency, can be critical in determining time-averaged stomatal conductance under unstable environmental conditions.

Optimal acclimation of the C3 photosynthetic system under enhanced CO2.

A range of studies of C3 plants have shown that there is a change in both the carbon flux and the pattern of nitrogen allocation when plants are grown under enhanced CO2. This paper examines evidence that allocation of nitrogen both to and within the photosynthetic system is optimised with respect to the carbon flux. A model is developed which predicts the optimal relative allocation of nitrogen to key enzymes of the photosynthetic system as a function of CO2 concentration. It is shown that evidence from flux control analysis is broadly consistent with this model, although at high nitrogen and under certain conditions at low nitrogen experimental data are not consistent with the model. Acclimation to enhanced CO2 is also assessed in terms of resource allocation between photosynthate sources and sinks. A means of assessing the optimisation of this source-sink allocation is proposed, and several studies are examined within this framework. It is concluded that C3 plants probably possess the genetic feedback mechanisms required to efficiently 'smooth out" any imbalance within the photosynthetic system caused by a rise in atmospheric CO2.

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation).

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.

Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO(2) Exchange.

The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO(2) assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light-mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed.

Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves.

An assay for the activity of ribulose bisphosphate carboxylase (Rubisco) activase in crude leaf extracts was developed. The assay is based on a spectrophotometric assay of Rubisco, and activase activity (in nanomoles activated Rubisco per minute per milligram chlorophyll) was calculated from the rate of increase in Rubisco activity over time. Activase activity measurements were made using samples from spinach (Spinacia oleracea) leaves undergoing (a) steady-state photosynthesis at various photon flux density (PFD) values and (b) nonsteady-state photosynthesis following an increase from darkness to a high PFD. Analysis of these samples showed that steady-state Rubisco activase activity was relatively low in darkness, increased with PFD, and saturated below 300 micromoles per square meter per second. Rubisco activity (measured spectrophotometrically) was also found to be low in darkness and to increase with PFD, but it saturated at much higher PFD values (approximately 1000 micromoles per square meter per second) along with the rate of photosynthesis. Following an increase in PFD from darkness to 650 micromoles per square meter per second, activase activity increased more or less linearly over a period of 5 to 6 minutes, after which it was constant. Rubisco activity, however, increased more slowly. The light-dependence of Rubisco activase is consistent with previous gas-exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD.

Distribution and accumulation of ultraviolet-radiation-absorbing compounds in leaves of tropical mangroves.

Ultraviolet (UV)-absorbing phenolic compounds that have been shown to be protective against the damaging: effects of UV-B radiation (Tevini et al., 1991, Photochem. Photobiol. 53, 329-333) were found in the leaf epidermis of tropical mangrove tree species. These UV-absorbing phenolic compounds and leaf succulence function as selective filters, removing short and energetic wavelengths. A field survey showed that the concentration of UV-absorbing compounds varied between species, between sites that would be experiencing similar levels of UV radiation, and between sun and shade leaves. Sun leaves have greater contents of phenolic compounds than shade leaves, and more saline sites have plants with greater levels in their leaves than less saline sites. In addition, increases in leaf nitrogen contents and quantum yields did not correlate with increasing levels of UV-absorbing compounds. It was concluded from these results that although UV-absorbing compounds form a UV-screen in the epidermis of mangrove leaves, UV radiation may not be the only factor influencing the accumulation of phenolic compounds, thus an experiment which altered the level of UV radiation incident on mangrove species was done. Near ambient levels of UVA and UV-B radiation resulted in a greater content of UV-absorbing compounds in Bruguiera parviflora (Roxb.) Wight and Arn. ex Griff., but did not result in increases in B. gymnorrhiza (L.) Lamk or Rhizophora apiculata Blume. Total chlorophyll contents were lower in R. apiculata when it was grown under near-ambient levels of UV radiation than when it was grown under conditions of UV-A and UV-B depletion, but no differences were observed between the UV radiation treatments in the other two species. There was no difference in leaf morphology, carotenoid/chlorophyll ratios, or chlorophyll a/b ratios between UV treatments, although these varied among species; B. parviflora had the highest carotenoid/chlorophyll ratio and R. apiculata had the lowest. Thus it is proposed that differences in species response tu UV radiation may be influenced by their ability to dissipate excess visible solar radiation.

Nonsteady-State Photosynthesis following an Increase in Photon Flux Density (PFD) : Effects of Magnitude and Duration of Initial PFD.

The response of photosynthesis to an increase in photon flux density (PFD) from low to higher PFD was investigated using spinach (Spinacia oleracea L.). The time-course for this response was qualitatively similar to that observed for a dark-to-high-PFD transition, showing an initial, rapid increase in photosynthesis over the first minute or so, followed by a slower increase lasting 5 to 10 minutes. This slow increase was approximately exponential and could be linearized using a semilogarithmic plot. The relaxation time (tau) for this slow phase was found to be a function of the starting PFD value. At starting PFD values below approximately 135 micromoles per square meter per second (including darkness), tau for the slow phase was approximately twice that observed for starting PFD values above 135 micromoles per square meter per second. This indicates a slower approach to steady state for leaves starting at PFD values below this threshold and a greater loss of potential photosynthesis. tau was relatively insensitive to starting PFD values below or above this transition value. The contribution of the slow phase to the total increase in photosynthesis following a low-to-high-PFD transition increased approximately exponentially with time at the lower PFD. The tau for the increase in the contribution of slow phase was determined to be 10.1 minutes. The implications of these data for activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase and for the functioning of the leaf in a fluctuating light environment are discussed.

Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.

Calcium binding by spinach stromal proteins.

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90-155 nmol·mg(-1) protein with "average" binding constants of 1.1-2.7·mM(-1). Both Mg(2+) and La(3+) inhibited calcium binding competitively, with "average" inhibitor constants of 0.26·mM(-1) and 39.4·mM(-1), respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg(-1) protein, but in an increase of the "average" affinity from 2.7 to 4.5·mM(-1). Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334-346) for binding site I of washed thylakoid membranes, showed that the free-Ca(2+) concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.

Substrate specificities of the enzymes of the oleate desaturase system from photosynthetic tissue.

In the microsomal fraction from young pea (Pisum sativum L.) leaves, the oleoyl moieties from oleoyl-CoA are principally transferred to the sn-2 position of phosphatidylcholine by oleoyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase. The major product of this acyl transfer is 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine. The 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine is subsequently converted into 1-palmitoyl(stearoyl)-2-linoleoyl phosphatidylcholine by the oleate desaturase complex without equilibrating with the bulk membrane phosphatidylcholine pool. Hence, both the acyl transfer to phosphatidylcholine and the subsequent desaturation of oleoyl moieties occur on the sn-2 position of phosphatidylcholine, and there is also a functional coupling of the acyltransferase and oleate desaturase.

The effects of Triton X-100 and n-octyl beta-D-glucopyranoside on energy transfer in photosynthetic membranes.

The effects of the non-ionic detergents Triton X-100 and n-octyl beta-D-glucopyranoside on energy transfer between pigment-protein complexes of Pisum sativum thylakoids were investigated. This was done by monitoring the 77K fluorescence-emission characteristics of stacked and unstacked thylakoids exposed to a range of detergent concentrations. At sub-critical micellar concentrations, the detergents had little effect, whereas above these concentrations they caused increases of up to 20-fold in short-wavelength fluorescence intensity and a shift in its maximum wavelength from 685 to 680 nm. Fluorescence-emission intensities at 695 and 735 nm were relatively unaffected by detergent treatments, although Triton X-100 caused a wavelength shift in the emission peak from 735 to 728 nm. The results are discussed in terms of reversible dissociation of pigment-protein complexes induced by mild detergent solubilization and the consequent cessation of inter-complex energy transfer.

Solubilization, purification and kinetic properties of three membrane-bound long-chain acyl-coenzyme-A thioesterases from microsomes of photosynthetic tissue.

Microsomal membranes of developing pea (Pisum sativum) leaves contained almost one third of the total long-chain acyl-CoA thioesterase activity found in the leaf cell. Three distinct forms of long-chain acyl-CoA thioesterase were purified by a combination of cholate-solubilization, dialysis, ion-exchange, and gel-filtration chromatography. Purification factors of 4600, 100 and 280 were achieved for the thioesterase forms I, II and III, respectively. Apparent molecular masses were: form I, 28 kDa; form II, 140 kDa; form III, 139 kDa. All the three thioesterases showed overlapping specificities towards palmitoyl-CoA, stearoyl-CoA, and oleoyl-CoA but were inactive towards short-chain acyl-CoAs, such as acetyl-CoA and malonyl-CoA. Each thioesterase exhibited complex kinetic behavior, which was consistent with differential affinities of the enzymes for monomeric and micellar forms of their substrates. The significance of the kinetic behavior and possible regulatory role of these enzymes are discussed.