Abscisic acid metabolism and episodic growth in cocoa

Abscisic acid (ABA) levels and metabolism were investigated in relation to shoot growth in cocoa (Theobroma cacao L.). ABA levels were high (14 nmoles/g fwt) in young flush leaves during shoot growth but gradually declined during the subsequent dormant period. ABA levels were low (1–2 nmoles/g fwt) in mature leaves when the terminal bud re-initiated growth. ABA-glucose ester (ABA-glu) levels were low (3–4 nmoles/g fwt) in the flush leaves during shoot growth and dormancy, however, ABA-glu levels increased more than 7-fold in these same leaves during the next flush cycle. ABA-glu levels then dropped significantly during the dormant period. Radiolabeled-ABA was metabolized to three products in cocoa leaves: ABA-glu, phaseic acid and dihydrophaseic acid. Catabolism of radiolabeled ABA was significantly greater in mature leaves during the dormant period when endogenous levels of ABA were high as compared to the period of active shoot growth when endogenous levels of ABA in mature leaves were low.     

Distribution of [14C]-labeled photosynthates within intensively cultured Populus clones during the establishment year

Seasonal patterns of [¹⁴C]-labeled photosynthate distribution within two intensively cultured Populus clones with contrasting phenology ( P. tristis × P. balsamifera cv. 'Tristis no. 1'; P. × euramericana cv. Eugenei) were investigated during the establishment year. During active shoot elongation upper mature leaves exported ¹⁴C acropetally to the expanding leaves and elongating internodes, and basipetally to the stem. Little ¹⁴C was exported to lower mature leaves or lateral branches. At budset the ¹⁴C export pattern shifted dramatically in the basipetal direction, i.e., to the lower stem, hardwood cutting, and roots. The timing of budset was the primary factor determining the differences between the clones, except that in all cases Tristis exported more ¹⁴C to the roots than Eugenei. After budset lower mature leaves had a similar export pattern to upper leaves, but the quantity of ¹⁴C exported to the roots was slightly higher. The results confirm the importance of autumn foliage for root growth in poplar. Clonal differences in seasonal patterns of photosynthate distribution offer potential for the poplar breeder seeking to match a clone's growth pattern with the specific growing season of the site.     

Seasonal and annual changes in leaf δ13C in two co-occurring Mediterranean oaks: relations to leaf growth and drought progression

1. Changes of δ¹³C and its relation to leaf development, biochemical content and water stress were monitored over a 2 year period in two co-occurring Mediterranean oak species: the deciduous Quercus pubescens and the evergreen Quercus ilex .
2. The time course of leaf δ¹³C showed different patterns in the two species. Young Q. pubescens leaves had a high δ¹³C and a marked decrease occurred during leaf growth. In contrast, leaves at budburst and maturity did not differ significantly in the case of Q. ilex . We suggest that the difference between δ¹³C of young leaves was linked to differential use of reserves of carbon compounds in the two species.
3. δ¹³C values of mature leaves were negatively correlated with minimum seasonal values of predawn water potential, suggesting that a functional adjustment to water resources occurred.
4. There was a significant correlation between individual δ¹³C values for two successive years. This interannual dependence showed that δ¹³C rankings between trees were constant through time.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Culture of Shoot Apices of Theobroma cacao

Surface sterilized buds of young cocoa plants (Theobroma cacao L.) taken at particular stages of the flush cycle were placed in Linsmaier and Skoog agar medium supplemented with a range of growth regulators. Only buds taken at the 1–2 (dormant) stage of the flush cycle and treated with gibberellic acid (GA₃) alone and GA₃plus kinetin (KN) supplement showed bud opening. In liquid Linsmaier and Skoog medium buds isolated at the 1–2 stage also responded to GA₃ and KN. In this case addition of KN caused bud opening, while GA₃ either initiated bud opening only or opening followed by leaf expansion depending on the concentration of GA₃ supplied. Bud development was inhibited when ABA was included in the medium hut this was overcome by the presence of GA₃ but not KN. Since a hormonal supplement was required for any response from the excised buds, it is suggested that the intermittent growth of the shoot apex in the intact plant may be determined by hormonal stimuli derived from other parts of the plant. The findings also indicate that the bud apices could be maintained in culture for long periods which may provide a basis for the development of a micropropagation procedure for cocoa.     

Variations in the amino acid composition of xylem sap of Betula pendula Roth. trees due to remobilization of stored N in the spring

Seasonal patterns of N translocation in the xylem sap of Betula pendula were studied, to determine whether specific amino acids were recovered in spring as a consequence of N remobilization. Seedlings were grown in sand culture and provided with ¹⁵NH₄¹⁵NO₃ (at 2·2 atom percent excess) for one growing season. The following winter dormant trees were transplanted into fresh sand and given N at natural abundance thereafter. Destructive harvests were taken during bud burst and leaf growth to determine the pattern of ¹⁵N remobilization and N uptake, along with isolation of xylem sap for analysis of their amino acid profiles and ¹⁵N enrichment by GC-MS. ¹⁵N remobilization occurred immediately following bud burst, while N derived from root uptake did not appear in the leaves until 12 d after bud burst. During N remobilization there was a 10-fold increase in the concentration of N in the xylem sap, due predominantly to increases in citrulline and glutamine. The ¹⁵N enrichment of these two amino acids demonstrated the increase in their concentration in the xylem sap following bud burst was due to N remobilization. These results are discussed in relation to measuring N remobilization and storage capacity of trees in the field.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

Altered resource allocation during seed development in Arabidopsis caused by the abi3 mutation

The regulation of whole-plant resource allocation during seed development in Arabidopsis thaliana was investigated by examining growth rate and partitioning of ¹⁴CO₂ in wild-type plants and those carrying the abi3 mutation. Plants carrying the abi3 mutation partitioned more resources into seed development than the wild type. The extra resources were available as a result of delayed senescence of the cauline leaves in the mutant. After supply of ¹⁴CO₂ at later stages of reproductive development differences in patterns of ¹⁴C distribution between mutant and wild type were consistent with long-term changes in growth and allocation. The role of long-distance signals in the regulation of seed yield in Arabidopsis is discussed.     

Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee ( Coffea arabica ) plants. [³H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide and [¹⁴C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 µmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [¹⁴C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [¹⁴C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [¹⁴C]nicotinamide in Camellia sinensis plants.     

Weather and climate controls over the seasonal carbon isotope dynamics of sugars from subalpine forest trees

We examined the environmental variables that influence the δ ¹³C value of needle and phloem sugars in trees in a subalpine forest. We collected sugars from Pinus contorta , Picea engelmannii and Abies lasiocarpa from 2006 to 2008. Phloem and needle sugars were enriched in ¹³C during the autumn, winter and early spring, but depleted during the growing season. We hypothesized that the late-winter and early-spring ¹³C enrichment was due to the mobilization of carbon assimilated the previous autumn; however, needle starch concentrations were completely exhausted by autumn, and we observed evidence of new starch production during episodic warm weather events during the winter and early-spring. Instead, we found that ¹³C enrichment was best explained by the occurrence of cold night-time temperatures. We also observed seasonal decoupling in the ¹³C/¹²C ratios of needle and phloem sugars. We hypothesized that this was due to seasonally-changing source-sink patterns, which drove carbon translocation from the needles towards the roots early in the season, before bud break, but from the roots towards the needles later in the season, after bud break. Overall, our results demonstrate that the ¹³C/¹²C ratio of recently-assimilated sugars can provide a sensitive record of the short-term coupling between climate and tree physiology.     

Diurnal and seasonal variation in the carbon isotope composition of leaf dark-respired CO2 in velvet mesquite (Prosopis velutina)

We evaluated diurnal and seasonal patterns of carbon isotope composition of leaf dark-respired CO₂ ( δ ¹³C_l) in the C₃ perennial shrub velvet mesquite ( Prosopis velutina ) across flood plain and upland savanna ecosystems in the south-western USA. δ ¹³C_l of darkened leaves increased to maximum values late during daytime periods and declined gradually over night-time periods to minimum values at pre-dawn. The magnitude of the diurnal shift in δ ¹³C_l was strongly influenced by seasonal and habitat-related differences in soil water availability and leaf surface vapour pressure deficit. δ ¹³C_l and the cumulative flux-weighted δ ¹³C value of photosynthates were positively correlated, suggesting that progressive ¹³C enrichment of the CO₂ evolved by darkened leaves during the daytime mainly resulted from short-term changes in photosynthetic ¹³C discrimination and associated shifts in the δ ¹³C signature of primary respiratory substrates. The ¹³C enrichment of dark-respired CO₂ relative to photosynthates across habitats and seasons was 4 to 6‰ at the end of the daytime period (1800 h), but progressively declined to 0‰ by pre-dawn (0300 h). The origin of night-time and daytime variations in δ ¹³C_l is discussed in terms of the carbon source(s) feeding respiration and the drought-induced changes in carbon metabolism.     

Carbon dioxide fixation in orchid aerial roots

Acidity fluctuation, CO₂ gas exchange, δ¹³C value, PEP carboxylase and RuBP carboxylase activities in aerial roots of selected thick-leaved orchid hybrids ( Arachnis and Aranthera ) were studied. Both aerial roots and leaves showed acidity fluctuation over a 24 h period. Dark acidification in aerial roots was enhanced at low temperature (15°C). Aerial roots had δ¹³C values close to those of leaves which have been previously demonstrated to possess crassulacean acid metabolism. Variation in δ¹³C values along the length of the roots was observed; the root tip having a less negative δ¹³C value (—13.34%‰) than the older portions of the roots (—14.55%‰). There was no net CO₂ fixation by aerial root, although ¹⁴³²CO₂ fixation was observed in light and in darkness. The pattern of fluctuation in activities of PEP carboxylase and RuBP carboxylase in aerial roots was similar to that obtained for the leaves. In both aerial roots and leaves, PEP carboxylase activity was several times higher than that of RuBP carboxylase.     

Translocation of 14C, carbohydrate content and activity of the enzymes of sucrose metabolism in rose petals temperatures

Both export of ¹⁴C from the source leaves of roses (Rosa × hybrida cv. Golden Times) and import of ¹⁴C to the petals were reduced by plant exposure to low night temperature. However, the import was affected to a greater extent than the export. During all stages of flower bud development the concentration of reducing sugars in petals of roses grown at reduced night temperature was lower than in petals of plants grown at higher night temperature. There was no significant difference in starch content in response to the night temperature, and the content of starch decreased toward complete flower bud opening. The concentration of sucrose in flowers at the low night temperature remained low during all stages of flower bud development, while at the high night temperature the concentration of sucrose increased during flower bud development, reaching a peak at the stage when petals start to unfold. At both temperatures the concentration of sucrose declined at complete flower opening. The activity of sucrose synthase (EC 2.4.1.14) was inhibited by low temperature in young rose shoots more than in the petals, while the activity of acid invertase (EC 3.2.1.26) was affected similarly in both tissues by the temperature treatments.     

Lymantria dispar herbivory induces rapid changes in carbon transport and partitioning in Populus nigra

We tested for rapid changes in photosynthate transport and partitioning in response to Lymantria dispar (L.) (Lepidoptera: Lymantriidae) (gypsy moth) herbivory in Populus nigra L. (Salicaceae). Transport and partitioning of [¹¹C]-photosynthate from young mature leaves were measured in vivo before and 18 h after leaf chewing by gypsy moth larvae, which were caged on three older leaves. Following herbivory, there was an increase in export speed of recently fixed carbon from younger mature leaves. The increased export speed was due to a quicker transit time of ¹¹C through the leaf, rather than a change in transport speed through the phloem. Additionally, basipetal partitioning of [¹¹C]-photosynthate was increased following herbivory. Neither of these changes was observed in control plants. This enhancement of export occurs even though herbivores are well known to induce increases in carbon allocation to secondary metabolites within leaves. Our results demonstrate that the use of non-destructive imaging of ¹¹C tracer is a powerful tool for examining plant responses to herbivory. Although the mechanisms underlying the rapid increase in carbon flux to stems and roots remain to be elucidated, our results raise the possibility of a coordinated whole plant response to herbivory. Thus, even when the herbivore specializes on only one plant tissue type, a whole plant approach may be key to understanding how plants respond to herbivory.     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Leaf δ13C reflects ecosystem patterns and responses of alpine plants to the environments on the Tibetan Plateau

Leaf δ¹³C is an indicator of water-use efficiency and provides useful information on the carbon and water balance of plants over longer periods. Variation in leaf δ¹³C between or within species is determined by plant physiological characteristics and environmental factors. We hypothesized that variation in leaf δ¹³C values among dominant species reflected ecosystem patterns controlled by large-scale environmental gradients, and that within-species variation indicates plant adaptability to environmental conditions. To test these hypotheses, we collected leaves of dominant species from six ecosystems across a horizontal vegetation transect on the Tibetan Plateau, as well as leaves of Kobresia pygmaea (herbaceous) throughout its distribution and leaves of two coniferous tree species ( Picea crassifolia, Abies fabri ) along an elevation gradient throughout their distribution in the Qilian Mountains and Gongga Mountains, respectively. Leaf δ¹³C of dominant species in the six ecosystems differed significantly, with values for evergreen coniferous13C values of the dominant species and of K. pygmaea were negatively correlated with annual precipitation along a water gradient, but leaf δ¹³C of A. fabri was not significantly correlated with precipitation in habitats without water-stress. This confirms that variation of δ¹³C between or within species reflects plant responses to environmental conditions. Leaf δ¹³C of the dominant species also reflected water patterns on the Tibetan Plateau, providing evidence that precipitation plays a primary role in controlling ecosystem changes from southeast to northwest on the Tibetan Plateau.     

Effect of UV-B radiation on the shoot dry matter production and stable carbon isotope composition of two Arabidopsis thaliana genotypes

An application of stable carbon isotope analysis to the mechanistic interpretation of ultraviolet-B (UV-B) effects on growth inhibition is described that is particularly useful for small plants such as Arabidopsis thaliana that are not well suited for gas exchange studies. Many investigators use tissue δ¹³C, relative abundance of ¹³C and ¹²C, as a proxy for water use efficiency and as an indicator of environmental effects on stomatal behaviour and on photosynthesis during growth. Discrimination against ¹³C is enhanced by both high stomatal conductance and damage to photosynthetic machinery. Because the thinning of the stratospheric ozone layer is permitting more UV-B to enter the biosphere, the mechanisms of action of UV-B radiation on plants are of particular current interest. Arabidopsis thaliana wild-type Landsberg erecta (L er ) and the UV-B-sensitive mutant fah I , deficient in UV-absorbing sinapate esters, were grown in a controlled environment and exposed to UV-B^BE doses of 0 or 6–7 kJ m⁻² day⁻¹. UV-B exposure decreased dry matter production and δ¹³C in both genotypes, but growth inhibition was generally greater in fah I than in L er . The fah I mutant also had less leaf greenness than L er . Changes in leaf tissue δ¹³C were detected before growth inhibition and were evident in treatments of both genotypes that did not cause marked growth effects. This suggests that the effects of UV-B contributing to increased carbon isotope discrimination in L er may have been primarily associated with high stomatal conductance, and in fah I with both high stomatal conductance and damage to photosynthetic machinery.     

Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [¹³C₆]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [¹³C₆]IAA, while [¹²C]IAA of endogenous origin formed a minor pool. However, the pool of [¹³C₆]IAA decreased from 90 to 80% of the total free IAA pool [¹³C₆]IAA+[¹²C]IAA) while being transported down the shoot. This dilution with [¹²C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.