Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.

     

Comparative Distribution and Metabolism of Xylem-Borne Amino Compounds and Sucrose in Shoots of Populus deltoides

The transport and metabolism of xylem-borne amino compounds and sucrose were investigated in rapidly growing shoots of cottonwood (Populus deltoides Bartr. ex Marsh.). ¹⁴C-labeled glutamine, threonine, alanine, glutamic acid, aspartic acid, and sucrose were applied to the base of severed stems for transport in xylem. Distribution and metabolism of the compounds were followed with autoradiography, microautoradiography, and radioassay. Three utilization patterns were observed: (a) little alanine and sucrose was transported to the laminae of either mature leaves or developing leaves. These compounds were taken up from xylem free-space and utilized in adjacent tissue; (b) threonine also did not move into mature leaves but was translocated to developing leaves or utilized in the stem; (c) glutamic acid and aspartic acid were transported directly into the laminae of mature leaves via the xylem. Relatively less ¹⁴C was retained in stems compared to the other compounds.

Metabolism of the test compounds also differed considerably. ¹⁴C from amino acids moved primarily into organic acids and protein. The ¹⁴C from sucrose was widely distributed among the chemical fractions, with a high percentage found in structural carbohydrates. Clearly, cottonwood stems contain efficient uptake and transfer systems that differentiate among various compounds moving from root to shoot in xylem.

     

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.     

Influence of internal sugar levels on apoplasmic retrieval of exogenous sucrose in source leaf tissue

Sugar levels in Beta vulgaris leaves were increased by heat-girdling the petiole and returning the plant to the controlled-environment chamber for 10 and 34 hours. After 10 hours, sucrose influx into the treated leaves was similar to the controls, although sucrose levels increased from 2.1 to 5.3 micromoles per milligram chlorophyll. However, after a 34-hour treatment, sucrose levels increased from 2.1 to 11.5 micromoles per milligram chlorophyll. In this instance, sucrose influx decreased relative to the untreated controls. Decreasing sugar levels by DCMU treatment resulted in a small stimulation of sucrose influx. A similar DCMU treatment applied to leaves of Allium cepa also resulted in an increase in sucrose influx. However, in A. cepa we could not attribute this increase to a lowering of sugar levels, as the kinetic profiles obtained from control leaves did not vary from each other throughout the day, despite considerable changes in sugar levels. Additionally, it appeared that sucrose uptake in onion may be set at some point and remains invariant throughout the day. Similar studies were also conducted on discs cut from mature leaves of Spinacia oleracea var America. Between 1 and 8 hours after the onset of the photoperiod, the sucrose content of the spinach leaves increased from 2.6 to 9.3 micromoles per milligram chlorophyll. A comparison of the kinetic profiles obtained from leaf discs, taken at these times, indicated that sucrose uptake was not influenced by these changes in internal sugar levels. The relationship between the above findings and `trans' inhibition of exogenous sucrose uptake is discussed. Although intermediate changes in sugar levels in sugar beet leaves did not appear to affect sucrose influx, autoradiographic studies revealed that these changes dramatically affected the partitioning of exogenously supplied [¹⁴C]sucrose. Our results indicate that while intermediate changes in internal sugar levels have little effect on sucrose influx across the plasmalemma, they may dramatically affect partitioning between the phloem and the mesophyll vacuole.     

Carbon partitioning and export from mature cotton leaves

The partitioning of carbon in intact, mature cotton (Gossypium hirsutum L.) leaves was examined by steady-state ¹⁴CO₂ labeling. Plants were exposed to dark periods of varying lengths, followed by similar illuminated labeling periods. These treatments produced leaves with a range of starch and soluble sugar contents, carbon exchange, and carbon export rates. Export during the illuminated periods was neither highly correlated with photosynthesis nor was export during the illuminated periods significantly different among the treatments. In contrast, the rate of subsequent nocturnal carbon export from these leaves varied widely and was found to be highly correlated with leaf starch content at the end of the illumination period (r = 0.934) and with nocturnal leaf respiration (r = 0.954). Leaves which had accumulated the highest levels of starch (about 275 micrograms per square centimeter) by the end of the illumination period exhibited nocturnal export rates very similar to those during the daylight hours. Leaves which accumulated starch to only 50 to 75 micrograms per square centimeter virtually ceased nocturnal carbon export. For leaves with starch accumulations of between 50 and 275 micrograms per square centimeter, nocturnal export was directly proportional to leaf starch at the end of the illumination period. After the nocturnal export rate was established, it continued at a constant rate throughout the night even though leaf starch and sucrose contents declined.     

Isolation and in vitro translation of polysomes from mature rye leaves

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.     

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.)

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in ¹⁴CO₂ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.     

Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars

In the present study, leaves of different plant species were girdled by the hot wax collar method to prevent export of assimilates. Photosynthetic activity of girdled and control leaves was evaluated 3 to 7 days later by two methods: (a) carbon exchange rate (CER) of attached leaves was determined under ambient CO₂ concentrations using a closed gas system, and (b) maximum photosynthetic capacity (A_max) was determined under 3% CO₂ with a leaf disc O₂ electrode. Starch, hexoses, and sucrose were determined enzymically. Typical starch storers like soybean (Glycine max L.) (up to 87.5 milligrams of starch per square decimeter in girdled leaves), cotton (Gossypium hirsutum L.), and cucumber (Cucumis sativus L.) responded to 7 days of girdling by increased (80-100%) stomatal resistance (r_s) and decreased A_max (>50%). On the other hand, spinach (Spinacia oleracea L.), a typical sucrose storer (up to 160 milligrams of sucrose per square decimeter in girdled leaves), showed only a slight reduction in CER and almost no change in A_max. Intermediate plants like tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuus L.), broad bean (Vicia faba L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.), which upon girdling store both starch and sucrose, responded to the girdle by a considerable reduction in CER but only moderate inhibition of A_max, indicating that the observed reduction in CER was primarily a stomatal response. Both the wild-type tobacco (Nicotiana sylvestris) (which upon girdling stored starch and hexoses) and the starchless mutant (which stored only hexoses, up to 90 milligrams per square decimeter) showed 90 to 100% inhibition of CER and approximately 50% inhibition of A_max. In general, excised leaves (6 days) behaved like girdled leaves of the respective species, showing 50% reduction of A_max in wild-type and starchless N. sylvestris but only slight decline of A_max in spinach. The results of the present study demonstrate the possibility of the occurrence of end-product inhibition of photosynthesis in a large number of crop plants. The long-term inhibition of photosynthesis in girdled leaves is not confined to stomatal responses since the A_max declined up to 50%. The inhibition of A_max by girdling was strongest in starch storers, but starch itself cannot be directly responsible, because the starchless mutant of N. sylvestris was also strongly inhibited. Similarly, the inhibition cannot be attributed to hexose sugars either, because soybean, cotton, and cucumber are among the plants most strongly inhibited although they do not maintain a large hexose pool. Spinach, a sucrose storer, showed the least inhibition in both girdled and excised leaf systems, which indicates that sucrose is probably not directly responsible for the end-product inhibition of photosynthesis. The occurrence of strong end-product inhibition appears to be correlated with high acid-invertase activity in fully expanded leaves. The inhibition may be related to the nature of soluble sugar metabolism in the extrachloroplastic compartment and may be caused by a metabolite that has different rates of accumulation and turnover in sucrose storers and other plants.     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Influence of mannose on the apoplasmic retrieval systems of source leaves

Experiments were conducted in which d-mannose was supplied to mature Beta vulgaris L. (sugar beet) leaves, via the transpiration stream, to perturb photosynthetic carbon allocation by sequestering cytosolic Pi. Biochemical and enzymic analyses conducted on this tissue indicated that mannose 6-P was present, that it was only slowly metabolized, and that after a 24-hour pretreatment sugar metabolism was slightly perturbed. However, sucrose retrieval by the mesophyll tissue was greatly impaired in 24-hour mannose-pretreated tissue, a response which was due in part to mannose acting as an osmoticum. Inhibition of glucose, fructose, and arginine uptake into mannose-treated sugar beet leaf discs indicated that mannose may elicit a general perturbation of all membrane transport processes. This conclusion was supported by our finding that sucrose efflux was increased from mannose-treated tissue. Analysis of adenine nucleotide levels showed that whereas these levels declined over the first 3 to 6 hours of the mannose treatment, by 24 hours they had recovered to near control values. Similar experiments conducted on Nicotiana rustica indicated that whereas mannose 6-P was present in mature leaves, it remained at a much lower level than that found in sugar beet. Sucrose uptake into N. rustica was insensitive to mannose pretreatment. However, glucosamine treatment, which is also thought to sequester cytosolic Pi, inhibited sucrose uptake in both N. rustica and B. vulgaris. Further, experiments conducted on N. tabacum L. var Xanthii showed that mannose caused an inhibition of sucrose uptake, indicating that a range of sensitivity to mannose exists between closely related species. These results are discussed in terms of possible mechanisms of inhibition.     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization

The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1′-deoxy-1′-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [¹⁴C]Sucrose and [¹⁴C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [¹⁴C]sucrose was supplied than when [¹⁴C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.     

Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection

We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo.     

Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants

The effect of CN⁻ and N₂ on the electrical membrane potential (E_m) was compared with that of CN⁻ on the ATP levels in cotyledons of Gossypium hirsutum and in Lemna gibba L. In mature cotton tissue, CN⁻ depolarized E_m to the energy-independent diffusion potential (E_D) in the dark. In the light E_m recovered transiently. The same was observed in leaves of Nicotiana, Avena, Impatiens, Kalanchoë, and in Lemna. In contrast, in young cotton cotyledons and tobacco leaves and, to a large extent, in +sucrose-grown Lemna, E_m was depolarized to E_D also in the light in a similar way as in the dark.

In Lemna grown without sucrose, the energy-dependent component of E_m was only partially depolarized by CN⁻ in dark or light. Cyanide plus salicylhydroxamic acid completely reduced E_m to E_D, abolished respiration and photosynthesis, and severely diminished the ATP level. This suggests the operation of a CN⁻-insensitive respiration in uninjured Lemna. The initial CN⁻-induced decay of the ATP level in cotton and Lemna was more rapid than the decay of E_m. CN⁻-induced oscillations of the ATP level were followed by similar but slower oscillations of E_m. This supports the view of a general dependence of E_m on ATP. Discrepancies between inhibitor-induced changes of E_m and ATP levels are suggested to result from additional regulation of E_m by the cytoplasmatic pH value.

A comparison of E_D in young and mature cotton cotyledons in the dark and in the light suggests that in growing young cotyledons the different effect of CN⁻ in the light is due to a less effective photosynthesis together with high mitochondrial respiration. In Lemna and in mature cotton tissue, E_m in the light is maintained by noncyclic photophosphorylation and photosystem II, which is only partly inhibited by CN⁻, thus resulting in an incomplete depolarization and recovery of E_m. Complete inhibition of photosynthetic O₂ evolution and membrane depolarization by CN⁻ plus salicylhydroxamic acid are suggested to result from photooxidation.

     

Changes in cotton leaf chemistry induced by volatile elicitors

Cotton (Gossypium hirsutum) leaves were exposed for 7 days to volatile chemicals originating from Aspergillus flavus-infected cotton leaves, A. flavus cultures or mechanically damaged cotton leaves. Volatiles from A. flavus-infected leaves triggered significant increases of 52 and 34% in phloroglucinol-reactive compounds in wounded or undamaged cotton leaves, respectively. Increased production of heliocides (C₂₅ terpenoid aldehydes) were found in the volatile recepient wounded or undamaged cotton leaves. The heliocides are natural insecticides presumed localized in the subepidermal pigment glands in leaves. Myrcene, a volatile precursor of heliocide H₂, also caused significant increases in heliocide production when leaves were exposed to the volatilized chemical.