Alterations in Growth, Photosynthesis, and Respiration in a Starchless Mutant of Arabidopsis thaliana (L.) Deficient in Chloroplast Phosphoglucomutase Activity

A mutant of Arabidopsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. The starchless phenotype, confirmed by quantitative enzymic analysis, is caused by a single recessive nuclear mutation which results in a deficiency of the chloroplast isozyme of phosphoglucomutase. When grown in a 12-h photoperiod, leaves of the wild-type accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions, the mutant accumulated relatively high levels of soluble sugars. Rates of growth and net photosynthesis of the mutant and wild-type were indistinguishable when the plants were grown in constant illumination. However, in a short photoperiod, the growth of the mutant was severely impaired, the rate of photosynthesis was depressed relative to the wild-type, and the rate of dark respiration, which was high following the onset of darkness, exhibited an uncharacteristic decay throughout the dark period. The altered control of respiration by the mutant, which may be related to the relatively high levels of soluble carbohydrate that accumulate in the leaf and stem tissue, is believed to be partially responsible for the low growth rate of the mutant in short days. The depressed photosynthetic capacity of the mutant may also reflect a metabolic adaptation to the accumulation of high levels of soluble carbohydrate which mimics the effects of alterations in source/sink ratio. The activities of sucrose phosphate synthase and acid invertase are significantly higher in the mutant than in the wild-type whereas ADP-glucose pyrophosphorylase activity is lower. This suggests that the activities of these enzymes may be modulated in response to metabolite concentrations or flux through the pathways.     

The base of the leaf acts as a localized sink for photosynthate in mature barley leaves

The gradients in photosynthetic and carbohydrate metabolism which persist within the fully expanded second leaf of barley ( Hordeum vulgare ) were examined. Although all regions of the leaf blade were green and photosynthetically active, the basal 5 cm, representing approximately 20% of the leaf area, retained some characteristics of sink tissue. The leaf blade distal from the leaf sheath exhibited characteristics typical of source tissue; the activities of sucrolytic enzymes (invertase and sucrose synthase) were relatively low, whilst that of sucrose phosphate synthase was high. These regions of the leaf accumulated sucrose throughout the photoperiod and starch only in the second half of the photoperiod whilst hexose sugars remained low. By contrast the leaf blade proximal to the leaf sheath retained relatively high activities of sucrolytic enzymes (especially soluble, acid invertase) whilst sucrose phosphate synthase activity was low. Glucose, as well as sucrose, accumulated throughout the photoperiod. Although starch accumulated in the second half of the photoperiod, a basal level of starch was present throughout the photoperiod, by contrast with the rest of the leaf. The ¹⁴CO₂ feeding experiments indicated that a constant amount of photosynthate was partitioned towards starch in this region of the leaf irrespective of irradiance. These findings are interpreted as the base of the leaf blade acting as a localized sink for carbohydrate as a result of sucrose hydrolysis by acid invertase.     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Diurnal Pattern of Translocation and Carbohydrate Metabolism in Source Leaves of Beta vulgaris L

Transitions in carbohydrate metabolism and translocation rate were studied for evidence of control of export by the sugar beet (Beta vulgaris L. Klein E.) source leaf. Steady-state labeling was carried out for two consecutive 14-hour light periods and various quantities related to translocation were measured throughout two 24-hour periods. Starch accumulation following illumination was delayed. Near the end of the light period, starch stopped accumulating, whereas photosynthesis rate and sucrose level remained unchanged. At the beginning of the dark period there was a 75-minute delay before starch was mobilized. The rate of import to the developing sink leaves at night was similar to that during the day, whereas export decreased considerably at night.

Starch accumulation and degradation seemed to be initiated in response to the level of illumination. Cessation of starch accumulation before the end of the light period was initiated endogenously. Exogenous control appeared to be mediated by the level of sucrose in the source leaf while endogenous control seemed to be keyed to photoperiod or photosynthetic duration.

     

Changes in Nonstructural Carbohydrates in Different Parts of Soybean (Glycine max [L.] Merr.) Plants during a Light/Dark Cycle and in Extended Darkness

Diurnal patterns of nonstructural carbohydrate (starch, sucrose, and hexose sugars) concentration were characterized in different parts (leaves, petioles, stems, and roots) of vegetative soybean (Glycine max [L.] Merr.) plants. Pronounced changes in all carbohydrate pools were observed in all plant parts during the normal photosynthetic period; however, starch accumulation within leaves accounted for more than 80% of the nonstructural carbohydrate accumulated by the plant during the light period. Efficiency of utilization of starch and sucrose during the normal dark period differed among organs, with leaves being most efficient in mobilizing starch reserves and roots being most efficient in utilizing sucrose reserves. The vast majority (about 85%) of the whole plant carbohydrate reserves present at the end of the photosynthetic period were utilized during the normal dark period. Sink leaf expansion ceased in plants transferred to extended darkness and the cessation in leaf expansion corresponded with carbohydrate depletion in the subtending source leaf and the remainder of the plant. Collectively, the results indicated that under the conditions employed, leaves are the whole plant's primary source of carbon at night as well as during the day.     

Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Soluble acid invertase activity in leaves is independent of species differences in leaf carbohydrates, diurnal sugar profiles and paths of phloem loading

Leaf sucrose, starch, hexose and maximum extractable soluble acid invertase activity were compared throughout the day in source leaves of 13 plant species chosen for their putative phloem-loading type (apoplastic or symplastic). Four species which represent the different phloem-loading types (tomato, barley, maize and Fuchsia ) were studied in detail. Using this information we wished to determine whether a positive correlation between foliar carbohydrates and acid invertase activity exists in leaves from different species and, furthermore, whether this relationship is determined by phloem-loading type. Acid invertase activity was relatively constant throughout the day in all species. The extent of sucrose, hexose and starch accumulation and the sucrose: starch ratio measured at a given time were species-dependent. No correlations were found between foliar soluble acid invertase activity and the hexose, sucrose or starch content of the leaves in any of the species, regardless of phloem-loading type. The species examined could be divided into three distinct groups: (1) high sucrose, low invertase; (2) low sucrose, low invertase; and (3) low sucrose, high invertase. The absence of an inverse relationship between leaf sucrose, hexose or starch contents and endogenous soluble acid invertase suggests that this enzyme is not directly involved in carbon partitioning in leaves but serves an auxiliary function.     

Carbon Partitioning in Mature Leaves of Pepper: Effects of Daylength

Grange, R. 1. 1985. Carbon partitioning in mature leaves ofpepper: effects of daylength.—J. exp. Bot. 36: 1749–1759. The partitioning of recently fixed carbon has been examinedin mature pepper leaves grown in 6, 10 or 14 h photoperiodsat different irradiances chosen to give similar radiation integralsand in a 6 h photoperiod at the lowest of these irradiances.The partitioning of carbon into export, starch, sugars and respirationwas followed over the photopenod and the subsequent night ina mature leaf. The maximum export rate during the day (approximately 18 µgC cm^–2 leaf h^–1) was not significantly differentamong the treatments. Net photosynthesis rate was directly relatedto irradiance; the proportion of net photosynthesis exportedduring the day was 33% in 6-h days and 57% in 14-h days. Leafstarch accumulation (as a proportion of net photosynthesis rate)increased slightly when plants were grown in 6-h days. The remobilization of starch and sugars at night allowed exportrates to remain similar over 24 h when plants were grown in10-h or 14-h photoperiods. Leaves grown in 6-h days showed nosignificant changes in export rate during the first few hoursof night but exhausted their starch reserves during the nightand export rates declined. Sucrose and hexose levels decreased at the onset of darkness,but did not fall below 40 µg cm^–2 in plants grownin 10-h or 14-h photoperiods; when this level was reached after3–4 h of darkness, starch breakdown began. In leaves grownin both 6-h treatments, sucrose levels fell below 40 µgcm^–2 when starch reserves were depleted during the nightand the export rate decreased concurrently. The results are discussed in relation to the control of exportand starch metabolism in the leaf. Key words: Pepper, partitioning, daylength     

Effect of Restricted Root Growth on Carbohydrate Metabolism and Whole Plant Growth of Cucumis sativus L

The effects of varied rooting volumes on root growth and source leaf carbohydrate metabolism were studied in greenhouse-grown cucumber (Cucumis sativus L cv Calypso) plants. Plants were grown for 7 weeks in container volumes that ranged from 0.4 to 5.9 liters. Plants grown in the smaller containers exhibited less leaf expansion, lower root and shoot weight, and fewer lateral stems than plants grown in the 5.9 liter containers. Shoot/root ratio was not altered by the container volume, suggesting coordination of root and shoot growth due to rooting volume. Source leaf carbon exchange rates, assimilate export rates, and starch accumulation rates for plants grown in 0.4 liter containers were approximately one-half or less in comparison to those for plants grown in 5.9 liter containers. Starch concentrations per unit leaf area were maintained at high levels in source leaves of plants grown in 0.4 liter containers over the entire day/night cycle. Lower extractable galactinol synthase activities and higher galactinol concentrations occurred in leaves of plants grown in 0.4 liter container volumes. The reduced sink demand, induced by restricted root growth, may have led to increased starch concentrations and to a reduction in stachyose biosynthesis in cucumber source leaves.     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.     

Diel patterns of leaf C export and of main shoot growth for Flaveria linearis with altered leaf sucrose-starch partitioning

Diel C export from source leaves of two Flaveria linearis lines [85-1: high cytosolic fructose-1,6-bisphosphatase (cytFBPase) and 84-9: low cytFBPase] were estimated using three methods, including leaf steady-state (14)CO(2) labelling, leaf metabolite analysis, and leaf dry mass analysis in conjunction with leaf CO(2) exchange measurements. Synthesis and accumulation of starch during the daytime were much higher in 84-9. Relative (14)C-export (export as a % of photosynthesis) in the light was 36% higher in 85-1. The diel export patterns from (14)C-analyses correlated with those based on metabolite or dry weight/gas exchange analyses during the daytime, but not during the night. Night-time export estimated from (14)C-disappearance was 3.6 times lower than those estimated using the other methods. Even though the starch degradation at night was greater for 84-9, night-time export in 84-9 was similar to 85-1, since 84-9 showed both higher respiration and accumulation of soluble sugars (i.e. glucose) at night. Patterns of (14)C allocation to sink organs were also different in the two lines. Main stem growth was less in 84-9, being reduced most in the light when leaf export was lower relative to 85-1. Supplementation with sucrose for 1 h daily via the roots at a time when leaf export in 84-9 was low relative to 85-1 increased the stem growth rate of 84-9 to a level similar with that of 85-1. This study provides evidence that diel C availability predicted by source strength (e.g. C-export rate) influences main stem extension growth and the pattern of sink development in F. linearis.     

Starch Accumulation in Leaves of Potato (Solanum tuberosum L.) during the First 18 Days of Photoperiod Treatment

Potato (Solanum tuberosum L.) plants were grown under long days(LD) of 18 h before a subset of the plants was transferred to10-h photosynthetic periods with either a dark night (SD) oran 8-h dim photoperiod extension with incandescent lamps (DE).Temperature was constant at 21 °C. Leaves were sampled atthe beginning and end of the high density light period for starchanalyses. Potato leaves accumulated starch more rapidly underSD than under LD; and this difference continued after a secondmajor sink, the tuber, began to develop. Starch accumulationover 10 h in SD leaves was three times higher than in LD leaves,even after 17 d of treatment. By this time SD gave higher wholeplant relative growth rates than LD, and the tuber mass of SDplants exceeded 30% of their total plant biomass. The DE treatmentresulted in starch accumulation intermediate to the LD and SDtreatments. Genotypes likewise differed: the earlier genotype,more strongly induced to tuberize, had higher leaf starch accumulationthan the later genotype. The effects of photoperiod and genotypewere also present when potatoes were grown at 27 °C, a temperatureunfavourable for tuberization under LD. Thus the formation ofa strong tuber sink was consistently associated with more rapidleaf starch accumulation. Potato, Solanum tuberosum L., cv. Norchip, photoperiod, temperature, genotype, starch accumulation, partitionin     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

Soluble Sugars as the Carbohydrate Reserve for CAM in Pineapple Leaves : Implications for the Role of Pyrophosphate:6-Phosphofructokinase in Glycolysis

Neutral ethanol-soluble sugar pools serve as carbohydrate reserves for Crassulacean acid metabolism (CAM) in pineapple (Ananas comosus (L.) Merr.) leaves. Levels of neutral soluble sugars and glucans fluctuated reciprocally with concentrations of malic acid. Hexose loss from neutral soluble-sugar pools was sufficient to account for malic acid accumulation with about 95% of the required hexose accounted for by turnover of fructose and glucose pools. Hexose loss from starch or starch plus lower molecular weight glucan pools was insufficient to account for nocturnal accumulation of malic acid. The apparent maximum catalytic capacity of pyrophosphate:6-phosphofructokinase (PPi-PFK) at 15°C was about 16 times higher than the mean maximum rate of glycolysis that occurred to support malic acid accumulation in pineapple leaves at night and 12 times higher than the mean maximum rate of hexose turnover from all carbohydrate pools. The apparent maximum catalytic capacity of ATP-PFK at 15°C was about 70% of the activity required to account for the mean maximal rate of hexose turnover from all carbohydrate pools if turnover were completely via glycolysis, and marginally sufficient to account for mean maximal rates of acidification. Therefore, at low night temperatures conducive to CAM and under subsaturating substrate concentrations, PPi-PFK activity, but not ATP-PFK activity, would be sufficient to support the rate of glycolytic carbohydrate processing required for acid accumulation. These data for pineapple establish that there are at least two types of CAM plants with respect to the nature of the carbohydrate reserve utilized to support nighttime CO₂ accumulation. The data further indicate that the glycolytic carbohydrate processing that supports acidification proceeds in different subcellular compartments in plants utilizing different carbohydrate reserves.     

Phloem-specific expression of pyrophosphatase inhibits long distance transport of carbohydrates and amino acids in tobacco plants

As reported in a previous paper [Lerchl et al. (1995) Plant Cell, 7, 259–270], expression of Escherichia coli inorganic pyrophosphatase in the cytosol under the control of the phloem-specific rolC promoter from, Agobacterium rhizogenes results in decreased growth of transgenic tobacco plants. In this paper we investigate the effect of the phloem-specific expression of pyrophosphatase on phloem metabolism, and on plant growth and allocation. A small decrease in the hexose phosphate/UDP-glucose ratio, the ATP/ADP ratio and the respiration rate in the midribs of the transformants provides evidence Hint mobilization of sucrose via pyrophosphate-dependent reactions is necessary for phloem energy metabolism. The source leaves of the transformants had higher levels of carbohydrates and amino acids and a much higher glutamine/glutamate ratio than the wild type, showing that export was inhibited and that the growth inhibition was not due to a lack of photoas-similates or organic nitrogen in the leaves. The accumulation of photoassimilates was paralleled by a decrease in photosynthesis, chlorophyll content and ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity, a small increase in hexose phosphates and triose phosphates and a decrease in glycerate 3-phosphate in the source leaves. There was a decrease of soluble sugars and amino acids in sink leaves of the transformants. In sink leaves amino acids decreased more than carbohydrates and a decrease in the glutamine/ glutamate ratio was observed. This was accompanied by a large decrease of nitrate. Sugars and amino acids were also reduced in the root tips of the transformants. The carbohydrate /amino acid ratio decreased 5-fold in the root tips, indicating a particularly smile shortage of carbohydrates. Relatively high levels of sugars and amino acids in the basal regions of the root and the increase in sugars in the midrib indicate that there is also increased leakage of assimilates out of the phloem during long-distance transport. Metabolism is required to maintain phloem function along the transport route, as well as for the initial step of loading. The transformants showed decreased stem and root growth. The growth inhibition was largest in conditions allowing rapid growth of the wild type (high light and nitrogen supply).