Ecophysiology of phloem loading in source leaves

The nature of phloem loading of photosynthesis products – either symplastic or apoplastic – has been a matter of debate over the last two decades. This controversy was reconciled by proposing a multiprogrammed loading mechanism. Different modes of phloem loading were distinguished on the basis of the variety of plasmodesmatal connectivity between the minor vein elements. Physiological evidence for at least two phloem loading mechanisms as well as recent support for coincidence between plasmodesmatal connectivity and the loading mechanism is shortly reviewed. The present paper attempts to correlate the plasmodesmatal connectivity between sieve element/companion cell complex and the adjacent cells (the minor vein configuration) – and implicitly the associate phloem loading mechanisms – with different types of climate. The minor vein configuration is a family characteristic. This enables one to relate vein configuration with ecosystem using the family distribution over the globe. The uneven distribution of vein types between terrestrial ecosystems indicates that apoplastic phloem loading predominates in cold and dry climate zones. Projection of the minor vein configuration on the Takhtajan system of flowering plants suggests evolution from apoplastic to symplastic phloem loading. Accordingly, the distribution of minor vein configurations suggests that drought and temperature stress have led to the transformation of the ancient symplastic mode into the more advanced apoplastic mode of loading.     

Control of photosynthate partitioning in spinach leaves

Experiments were carried out to estimate the elasticity coefficients and thence the distribution of control of sucrose synthesis and photosynthate partitioning between cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase (SPS), by applying the dualmodulation method of Kacser and Burns (1979, Biochem. Soc. Trans. 7, 1149–1161). Leaf discs of spinach (Spinacia oleracea L.) were harvested at the beginning and end of the photoperiod and illuminated at five different irradiances to alter (i) the extent of feedback inhibition and (ii) the rate of photosynthesis. The rate of CO₂ fixation, sucrose synthesis and starch synthesis were measured and compared with the activation of SPS, and the levels of fructose-2,6-bisphosphate (Fru2,6bisP) and metabolites. Sucrose synthesis increased progressively with increasing irradiance, accompanied by relatively large changes of SPS activity and Fru2,6bisP, and relatively small changes of metabolites. At each irradiance, leaf discs harvested at the end of the photoperiod had (compared with leaf discs harvested at the beginning of the photoperiod) a decreased rate of sucrose synthesis, increased starch synthesis, decreased SPS activity, increased Fru2,6bisP, a relatively small (20%) increase of most metabolites, no change of the glycerate-3-phosphate: triose-phosphate ratio, a small increase of NADPmalate dehydrogenase activation, but no inhibition of photosynthesis. The changes of sucrose and starch synthesis were largest in low light, while the changes of SPS and Fru2,6bisP were as large, or even larger, in high light. It is discussed how these results provide evidence that the control of sucrose synthesis is shared between SPS and fructose-1,6-bisphosphatase, and provide information about the in-vivo response of these enzymes to changes in the levels of their substrates and effectors. At low fluxes, feedback regulation is very effective at altering partitioning. In high light, changes of SPS activation and Fru2,6bisP can be readily overriden by increasing levels of metabolites.     

Carbon metabolism in leaves of micropropagated sugarcane during acclimatization phase

The activity of the main enzymes related to the sucrose metabolism, photosynthesis, and sucrose concentration were studied in sugarcane (Saccharum spp hybrid) plantlets. Acclimatization was developed in two steps. (1) Light intensity of 1,000 μmol m⁻² s⁻¹ and 90% relative humidity during the first 21 d; followed by 2,000 μmol m⁻² s⁻¹ and approximately 80% of relative humidity. All measurements were carried out at the end of rooting phase concomitant with day 0 of acclimatization and at 7-d intervals thereafter (0, 7, 14, 21, 28, 35, 42 d). As the in vitro plantlets were transferred to the acclimatization phase, photosynthesis increased significantly during the first 7 d. After this period, the increase was constant with only a small but nonsignificant decline after being transferred to the uncontrolled external conditions. The activity of the sucrose synthase began to show a decrease, starting from day 7, and was related to the changes that began to happen in these plants from its adaptation to new ex vitro conditions. Due to the increase of fresh weight favored by the high light intensity and lower relative humidity, an increase of the sucrose phosphate synthase activity was observed. The maximum activity of the acid and neutral invertases was reached at 14 and 21 d, respectively, after 21 d of acclimatization period. There was a marked tendency for the activity of both enzymes to decrease. The sucrose content was decreased only in the first 7 d. The metabolism of sugarcane plantlets seemed to be susceptible to the environmental changes during the acclimatization phase but did not contribute to inhibitory factors for normal development.     

Phloem unloading following reactivation in predarkened mature maize leaves

Mature leaf blades of 48-h predarkened maize plants (Zea mays L. cv. Prior) were excised, and treated apically as the source (light, normal air) and basally as the sink (light or dark, air without CO₂). After providing the source portion with ¹⁴CO₂, the sink portions were harvested after 2, 7 or 14 h by freezing with liquid nitrogen, grinding, and freeze-drying. Extracts, fractionated by ionexchange resins into neutral, basic and acid fractions, were chromatographed on thin cellulose layers, and autoradiographed. Identification of labeled compounds was carried out by co-chromatography with authentic labeled substances. Activities of enzymes pertaining to the metabolism of sucrose were checked. Results show that the source supplies sucrose to the sink, where it is unloaded and metabolized by acid invertase (EC 3.2.1.26) in both the light and the dark. Starch appearing in the sink only in the light, after 7 h of re-illumination, yields labeled glucose upon hydrolysis. Although sucrose-phosphate synthetase (EC 2.4.1.14) is active in sinks and in isolated vascular-bundle fragments, it remains questionable whether sucrose unloaded from sieve tubes is metabolized by a method other than inversion. Sucrose synthetase (EC 2.4.1.13) was found to be inactive. Obviously, the main metabolite of unloaded sucrose is glucose-6-phosphate, giving access to the glycolytic pathway. The main difference between the sinks in the light and the dark is the lack of labeled glycine and serine in the dark. This indicates that in the light decarboxylation of glycine yields CO₂, which is recycled photosynthetically.Abbrevations Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - TLC thin-layer chromatography - UDPGlc uridine 5-diphosphate glucose     

Photoassimilate-transport characteristics of nonchlorophyllous and green tissue in variegated leaves of Coleus blumei Benth

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N₂ atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [¹⁴C]sucrose. Minor veins of green tissue in the same leaves accumulated [¹⁴C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of intermediary cells, large companion cells connected to the bundle sheath by abundant plasmodesmata.Abbreviation PCMBS p-chloromercuribenzenesulfonic acid     

The relationship between the activation state of sucrose-phosphate synthase and the rate of CO2 assimilation in spinach leaves

The relationship between the gas-exchange characteristics of spinach (Spinacia oleracea L.) leaves and the activation state of sucrose-phosphate synthase was examined at different intercellular partial pressures of CO₂ at two different photon flux densities. There was a strong positive correlation between the activation state of sucrose-phosphate synthase and the assimilation rate. The relationship was the same at both photon flux densities, indicating that the activation state of the enzyme is determined by a product of carbon assimilation, rather than directly by light.Abbreviations A assimilation rate for CO₂ - p _i intercellular CO₂pressure - PFD photon flux density - SPS sucrose-phosphate-synthase - Glc6P glucose-6-phosphate - Fru6P fructose-6-phosphate A.B. was the recipient of a visiting fellowship from the National Research Council of the Italy. This work was also supported by the Science and Engineering Research Council and the Agricultural and Food Research Council, UK.     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

Determination of GA3 in Chara vulgaris by capillary electrophoresis system

Method for simultaneous measurement of gibberellic acid was applied using capillary zone electrophoresis. Gibberellic acid was identified in extracts of apical part of thallus of Chara vulgaris L. The amount of gibberellins measured on the basis of activity determined by the micro-drop bioassay (59.8 mg·kg⁻¹; with gibberellic acid as a standard) was comparable with that estimated by capillary electrophoresis (54.9 mg·kg⁻¹).     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Peroxisomal enzyme activities in attached senescing leaves

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and -oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes.This work was supported by CNR Italy, special grant RAISA, subproject 2, paper no. 26.     

Intercellular compartmentation of sucrose synthesis in leaves of Zea mays L.

The incorporation of ¹⁴C into sucrose and hexose phosphates during steady-state photosynthesis was examined in intact leaves of Zea mays L. plants. The compartmentation of sucrose synthesis between the bundle sheath and mesophyll cells was determined by the rapid fractionation of the mesophyll and comparison of the labelled sucrose in this compartment with that in a complete leaf after homogenisation. From these experiments it was concluded that the majority of sucrose synthesis occurred in the mesophyll cell type (almost 100% when the time-course of sucrose synthesis was extrapolated to the time of ¹⁴C-pulsing). The distribution of enzymes involved in sucrose synthesis between the two cell types indicated that sucrose-phosphate synthetase was predominantly located in the mesophyll, as was cytosolic (neutral) fructose-1,6-bisphosphatase activity. Stromal (alkaline) fructose-1,6-bisphosphatase activity was found almost exclusively in the bundle-sheath cells. No starch was found in the mesophyll tissue. These data indicate that in Zea mays starch and sucrose synthesis are spatially, separated with sucrose synthesis occurring in the mesophyll compartment and starch synthesis in the bundle sheath.     

Reactivation of phloem export in mature maize leaves after a dark period

Mature leaves of corn plants (Zea mays L. cv. Prior) which were darkened for 48 h contain neither bundle-sheath starch nor glucose, and their sucrose content is below 5 M. In such leaves phloem export has ceased. When re-illuminated, photosynthetic sucrose production starts without delay, but the sucrose: glucose ratio is 1.25:1. Obviously, most of the new-formed sugar is utilized locally. Labeling with ¹⁴CO₂ has shown that phloen export starts 30 to 40 min after the onset of photosynthesis, when the sucrose: glucose ratio has increased to 13:1. The first newly formed starch can be detected when phloem export is reactivated. Glucose content remains constantly low af about 2 M for at least 2 h, and it never exceeds 10 M. Radioactivity in the exporting veins is about five times higher after 2 to 7 h of re-illumination than in the 14-h-day plant. Therefore, phloem export is either intensified during the period of reactivation or exported assimilates are partly unloaded along their way. Comparison of photosynthetic activity of equal-sized leaf strips has shown that both accumulation of photosynthates and radioactivity of exporting veins are about three times higher in the detached strip than in the strip which remained attached to the mother plant.     

Effect of plant growth substances on the growth of axillary buds in cultured stem segments of Phaseolus vulgaris L.

The hormonal control of axillary bud growth was investigated in cultured stem segments of Phaseolus vulgaris L. When the stem explants were excised and implanted with their apical end in a solid nutrient medium, outgrowth of the axillary buds-located at the midline of the segment-was induced. However, if indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) was included in the medium, bud growth was inhibited. The exposure of the apical end to IAA also caused bud abscission and prevented the appearance of new lateral buds.In contrast to apically inserted segments, those implanted in the control medium with their basal end showed much less bud growth. In these segments, the auxin added to the medium either had no effect or caused a slight stimulation of bud growth.The IAA transport inhibitor N-1-naphthylphthalamic acid (NPA) relieved bud growth inhibition by IAA. This suggests that the effect of IAA applied at the apical end requires the transport of IAA itself rather than a second factor. With the apical end of the segment inserted into the IAA-containing medium, simultaneous basal application of IAA relieved to some extent the inhibitory effect of the apical IAA treatment. These results, together with data presented in a related article [Lim R and Tamas I (1989) Plant Growth Regul 8: 151–164], show that the polarity of IAA transport is a critical factor in the control of axillary bud growth.Of the IAA conjugates tested for their effect on axillary bud growth, indoleacetyl alanine, indoleacetic acid ethyl ester, indoleacetyl-myo-inositol and indoleacetyl glucopyranose were strongly inhibitory when they were applied to the apical end of the stem explants. There was a modest reduction of growth by indoleacetyl glycine and indoleacetyl phenylalanine. Indoleacetyl aspartic acid and indoleglyoxylic acid had no effect.In addition to IAA and its conjugates, a number of other plant growth substances also affected axillary bud growth when applied to the apical end of stem segments. Myo-inositol caused some increase in the rate of growth, but it slightly enhanced the inhibitory effect of IAA when the two substances were added together. Gibberellic acid (GA₃) caused some stimulation of bud growth when the explants were from younger, rather than older plants. The presence of abscisic acid (ABA) in the medium had no effect on axillary bud growth. Both kinetin and zeatin caused some inhibition of axillary buds from younger plants but had the opposite effect on buds from older ones. Kinetin also enhanced the inhibitory effect of IAA when the two were applied together.In conclusion, axillary buds of cultured stem segments showed great sensitivity to auxins and certain other substances. Their growth responded to polarity effects and the interaction among different substances. Therefore, the use of cultured stem segments seems to offer a convenient, sensitive and versatile test system for the study of axillary bud growth regulation.     

Indole acetic acid mimics the effect of salt stress in relation to enzymes of carbohydrate metabolism in chickpea seedlings

The effect of addition of indole acetic acid (3 M) andNaCl (75 mM) on growth and enzymes of carbohydrate metabolism inchickpea seedlings was compared. In comparison with control seedlings, theseedlings growing in the presence of indole acetic acid (IAA) had reducedamylase activity in cotyledons and enhanced sucrose synthase (SS) and sucrosephosphate synthase (SPS) activities in cotyledons and shoots at all days ofseedling growth. Compared with control seedlings, sucrose content was higher incotyledons, shoots and roots and reducing sugar content was lower in shoots ofIAA treated seedlings. A low invertase (acid and alkaline) activity in shoots ofIAA treated seedlings could lead to reduced sink strength and hence decreasedgrowth of seedlings. Effects of NaCl stress on growth and activities of amylase,SS and SPS in cotyledons and invertase, SS and SPS in shoots were similar tothose observed with addition of IAA.     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential