Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting (14)C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.     

A bypass of sucrose synthase leads to low internal oxygen and impaired metabolic performance in growing potato tubers

Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-beta-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of (14)C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.     

Differential regulation of sorbitol and sucrose loading into the phloem of Plantago major in response to salt stress

Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major.     

AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.     

Cucumber mosaic virus infection affects sugar transport in melon plants

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed (14)CO(2) revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.     

Nonvascular,Symplasmic Diffusion of Sucrose Cannot Satisfy the Carbon Demands of Growth in the Primary Root Tip of Zea mays L

Nonvascular, symplasmic transport of sucrose (Suc) was investigated theoretically in the primary root tip of maize (Zea mays L. cv WF9 x Mo 17) seedlings. Symplasmic diffusion has been assumed to be the mechanism of transport of Suc to cells in the root apical meristem (R.T. Giaquinta, W. Lin, N.L. Sadler, V.R. Franceschi [1983] Plant Physiol 72: 362-367), which grow apical to the end of the phloem and must build all biomass with carbon supplied from the shoot or kernel. We derived an expression for the growth-sustaining Suc flux, which is the minimum longitudinal flux that would be required to meet the carbon demands of growth in the root apical meristem. We calculated this flux from data on root growth velocity, area, and biomass density, taking into account construction and maintenance respiration and the production of mucilage by the root cap. We then calculated the conductivity of the symplasmic pathway for diffusion, from anatomical data on cellular dimensions and the frequency and dimensions of plasmodesmata, and from two estimates of the diffusive conductance of a plasmodesma, derived from independent data. Then, the concentration gradients required to drive a growth-sustaining Suc flux by diffusion alone were calculated but were found not to be physiologically reasonable. We also calculated the hydraulic conductivity of the plasmodesmatal pathway and found that mass flow of Suc solution through plasmodesmata would also be insufficient, by itself, to satisfy the carbon demands of growth. However, much of the demand for water to cause cell expansion could be met by the water unloaded from the phloem while unloading Suc to satisfy the carbon demands of growth, and the hydraulic conductivity of plasmodesmata is high enough that much of that water could move symplasmically. Either our current understanding of plasmodesmatal ultrastructure and function is flawed, or alternative transport mechanisms must exist for Suc transport to the meristem.     

The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development

The sucrose (Suc) H(+)-cotransporter StSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. Reduced SUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc- and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm.     

Functional characterization of the Arabidopsis AtSUC2 Sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport

AtSUC2 (At1g22710) encodes a phloem-localized sucrose (Suc)/H(+) symporter necessary for efficient Suc transport from source tissues to sink tissues in Arabidopsis (Arabidopsis thaliana). AtSUC2 is highly expressed in the collection phloem of mature leaves, and its function in phloem loading is well established. AtSUC2, however, is also expressed strongly in the transport phloem, where its role is more ambiguous, and it has been implicated in mediating both efflux and retrieval to and from flanking tissues via the apoplast. To characterize the role of AtSUC2 in controlling carbon partitioning along the phloem path, AtSUC2 cDNA was expressed from tissue-specific promoters in an Atsuc2 mutant background. Suc transport in this mutant is highly compromised, as indicated by stunted growth and the accumulation of large quantities of sugar and starch in vegetative tissues. Expression of AtSUC2 cDNA from the 2-kb AtSUC2 promoter was sufficient to restore growth and carbon partitioning to nearly wild-type levels. The GALACTINOL SYNTHASE promoter of Cucumis melo (CmGAS1p) confers expression only in the minor veins of mature leaves, not in the transport phloem of larger leaf veins and stems. Mutant plants expressing AtSUC2 cDNA from CmGAS1p had intermediate growth and accumulated sugar and starch, but otherwise they had normal morphology. These characteristics support a role for AtSUC2 in retrieval but not efflux along the transport phloem and show that the only vital function of AtSUC2 in photoassimilate distribution is phloem loading. In addition, Atsuc2 mutant plants, although debilitated, do grow, and AtSUC2-independent modes of phloem transport are discussed, including an entirely symplastic pathway from mesophyll cells to sink tissues.     

Developmental regulation of anoxic stress tolerance in maize

Anoxia associated with flooding stress is detrimental to plant growth and productivity. When maize seedlings 2 to 7 d old were exposed to anoxic stress, 3-d-old seedlings were found to have much lower tolerance than 2-d-old seedlings. Ninety per cent of 2-d-old seedlings survived 72 h of anoxic stress compared with 0% of the 3-d-old seedlings. Since 2-d-old isolated root tips survived anoxic stress better than 3-d-old tips, the anoxic tolerance of 2-d-old seedlings was independent of the translocation of nutrient reserves from the endosperm to the root. The addition of glucose to the medium improved the anoxia tolerance of 2-d-old seedlings by 25% but had no effect on 3-d-old seedlings. Acclimation by pre-cxposure to 4% oxygen and pre-treatment with 100mmol m^?1 abscisic acid (ABA) improved the anoxia tolerance of 3-d-old seedlings by 2- and 4-fold, respectively. However, acclimation and ABA treatment had no effect on 2-d-old seedlings. The results indicate that anoxia tolerance in maize is develop-mentally regulated. The mechanism of anoxia tolerance innate to 2-d-old seedlings was inducible in 3-d-old seedlings by acclimation or treatment with ABA.     

UV-B response of green and etiolated barley seedlings

7-d-old etiolated and green barley seedlings (Hordeum vulgare L. cv. Alfa) were irradiated with UV-B for 30 min and then kept for 24 h in light or darkness. Chlorophyll (Chl) synthesis was inhibited by about 30 % as a result of UV-B irradiation, but there were no significant changes in photochemical activity measured by variable to maximum fluorescence ratio (F_v/F_m), quantum yield (Φ_PS2) and oxygen evolution rate. Electron transport of etiolated seedlings was similar to that of green ones, nevertheless, the Chl content was more then 2-fold lower. Ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits were diminished as a result of UV-B irradiation in etiolated and green plants, especially in those kept in the darkness. Catalase activity decreased and total superoxide dismutase activity increased in green and etiolated plants following UV-B treatment. When benzidine was used as a substrate, an isoform located between guaiacol peroxidases 2 and 3 (guaiacol peroxidase X) appeared, which was specific for UV-B treatment. As a result of irradiation, the contents of UV-B absorbing and UV-B induced compounds increased in green seedlings but not in etiolated seedlings.     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

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).     

The sucrose transporter of celery. Identification and expression during salt stress

In celery (Apium graveolens L.), long-distance transport of reduced carbon occurs both in the form of sucrose (Suc) and mannitol. The presence of mannitol has been related to the resistance of celery to salt stress. To investigate the transport events occurring during salt stress, we have cloned the H(+)/Suc transporter of celery AgSUT1 (A. graveolens Suc uptake transport 1) from a mature leaf cDNA library. The function of the encoded protein was confirmed by expression in yeast. AgSUT1 is a H(+)/Suc transporter with a high affinity for Suc (K(m) of 139 microM). Another closely related cDNA (AgSUT2) was also identified. AgSUT1 is mainly expressed in mature leaves and phloem of petioles, but also in sink organs such as roots. When celery plants were subjected to salt stress conditions (30 d watering with 300 mM NaCl) favoring mannitol accumulation (J.D. Everard, R. Gucci, S.C. Kann, J.A. Flore, W.H. Loescher [1994] Plant Physiol 106: 281-292), AgSUT1 expression was decreased in all organs, but markedly in roots. The results are discussed in relation to the physiology of celery.     

The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [(14)C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [(14)C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [(14)C]Suc converted to hexose falls dramatically. Instead, most of the [(14)C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.     

Phloem loading. A reevaluation of the relationship between plasmodesmatal frequencies and loading strategies

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.     

Transpiration rate. An important factor controlling the sucrose content of the guard cell apoplast of broad bean

Evaporation of water from the guard cell wall concentrates apoplastic solutes. We hypothesize that this phenomenon provides two mechanisms for responding to high transpiration rates. First, apoplastic abscisic acid is concentrated in the guard cell wall. Second, by accumulating in the guard cell wall, apoplastic sucrose (Suc) provides a direct osmotic feedback to guard cells. As a means of testing this second hypothesized mechanism, the guard cell Suc contents at a higher transpiration rate (60% relative humidity [RH]) were compared with those at a lower transpiration rate (90% RH) in broad bean (Vicia faba), an apoplastic phloem loader. In control plants (constant 60% RH), the guard cell apoplast Suc content increased from 97 +/- 81 femtomol (fmol) guard cell pair(-1) to 701 +/- 142 fmol guard cell pair(-1) between daybreak and midday. This increase is equivalent to approximately 150 mM external, which is sufficient to decrease stomatal aperture size. In plants that were shifted to 90% RH before daybreak, the guard cell apoplast Suc content did not increase during the day. In accordance, in plants that were shifted to 90% RH at midday, the guard cell apoplast Suc content declined to the daybreak value. Under all conditions, the guard cell symplast Suc content increased during the photoperiod, but the guard cell symplast Suc content was higher (836 +/- 33 fmol guard cell pair(-1)) in plants that were shifted to 90% RH. These results indicate that a high transpiration rate may result in a high guard cell apoplast Suc concentration, which diminishes stomatal aperture size.     

An Arabidopsis mutant impaired in coenzyme A biosynthesis is sugar dependent for seedling establishment

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4'-phosphopantothenoyl-cysteine decarboxilase that generates 4'-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in beta-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.