Mohammad Hajirezaei

Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning

Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (P(i) ), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high P(i) levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis-related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. P(i) acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, P(i) repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that P(i) acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis-associated phosphate transporters. The role of these effects in the suppression of symbiosis under high P(i) conditions is discussed.

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with “antisense” constructs to the genes encoding the α-and β-subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-14C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U14-C] sucrose. (vii) Metabolism (cycling) of [U-14C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-14C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-13C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Abstract. Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.

Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism.

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3–5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.

Cloning and Characterization of the Gene Cluster for Palatinose Metabolism from the Phytopathogenic Bacterium Erwinia rhapontici

Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

Host-related metabolic cues affect colonization strategies of a root endophyte

The mechanisms underpinning broad compatibility in root symbiosis are largely unexplored. The generalist root endophyte Piriformospora indica establishes long-lasting interactions with morphologically and biochemically different hosts, stimulating their growth, alleviating salt stress, and inducing local and systemic resistance to pathogens. Cytological studies and global investigations of fungal transcriptional responses to colonization of barley and Arabidopsis at different symbiotic stages identified host-dependent colonization strategies and host-specifically induced effector candidates. Here, we show that in Arabidopsis, P. indica establishes and maintains biotrophic nutrition within living epidermal cells, whereas in barley the symbiont undergoes a nutritional switch to saprotrophy that is associated with the production of secondary thinner hyphae in dead cortex cells. Consistent with a diversified trophic behavior and with the occurrence of nitrogen deficiency at the onset of saprotrophy in barley, fungal genes encoding hydrolytic enzymes and nutrient transporters were highly induced in this host but not in Arabidopsis. Silencing of the high-affinity ammonium transporter PiAMT1 gene, whose transcripts are accumulating during nitrogen starvation and in barley, resulted in enhanced colonization of this host, whereas it had no effect on the colonization of Arabidopsis. Increased levels of free amino acids and reduced enzymatic activity for the cell-death marker VPE (vacuolar-processing enzyme) in colonized barley roots coincided with an extended biotrophic lifestyle of P. indica upon silencing of PiAMT1. This suggests that PiAmt1 functions as a nitrogen sensor mediating the signal that triggers the in planta activation of the saprotrophic program. Thus, host-related metabolic cues affect the expression of P. indica’s alternative lifestyles.

Comparative analysis of abscisic acid content and starch degradation during storage of tubers harvested from different potato varieties

SummarySeveral lines of evidence suggest a prominent role of abscisic acid in controlling the length of the dormancy period of potato tubers. The ABA content was measured in tubers of six different potato varieties during storage. Consistent with previous publications a continuous decline in ABA content during storage was evident. However, this decline of ABA did not correlate with the sprouting behaviour of the tubers, ruling out that ABA is responsible for the break of dormancy. Activities of starch degrading and glycolytic enzymes were determined to investigate whether changes in their activities would correlate with the break of dormancy. Most of the enzymes investigated did not change significantly during storage. After the onset of sprouting activity of beta-amylase increased two-fold and alpha-amylase activity was stimulated by approximately 30%. The increase in beta-amylase activity was due to transcriptional activation, as shown by northern analysis. Since no change of starch degrading enzymes could be detected prior visible sprout growth, starch degradation is not a prerequisite for the initiation of sprouting.

Transgenic tobacco plants with strongly decreased expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase do not differ significantly from wild type in photosynthate partitioning, plant growth or their ability to cope with limiting phosphate, limiting nitrogen and suboptimal temperatures

Transformation of tobacco with the potato gene encoding the subunit of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) in the antisense orientation under the control of the constitutive CaMV 35S promoter, followed by selfing and crossing of the transformants, generated a line of tobacco (5–37) with up to an 85% reduction in PFP activity in the shoot. Transformants containing a sense construct (4-40-91) contained only 1–3% of wild-type PFP, presumably due to co-suppression. Rates of photosynthesis and partitioning between sucrose and starch in source leaves were identical in 4-40-91 transformants and the wild type. In the dark in sink leaves of 4-40-91 transformants, levels of hexose phosphates were up to 50% higher, glycerate-3-phosphate 30% lower and fructose-2,6-bisphosphate threefold higher than in the wild type; inorganic pyrophosphate, pyruvate and the ATP/ADP ratio were unaltered. Low -PFP and wild-type plants did not differ significantly in their rate of growth at 25° C and 200 μmol quanta · m−2 · s−1 on full nutrient medium. Growth on limiting phosphate and limiting nitrogen was inhibited identically in the wild type and transformants, and transformants adjusted their shoot/root ratio in an identical manner to the wild type. Differences in fructose-2,6-bisphosphate and glycolytic metabolites between the wild type and transformants were no larger in these suboptimal nutrient conditions, than in optimal conditions. Growth of the wild type and 4-40-91 transformants was inhibited identically at 12° C compared to 25° C. Differences in fructose-2,6-bisphosphate were smaller when the genotypes were compared at 12° C than at 25° C. We conclude that PFP does not play an essential role in photosynthate partitioning in source leaves. During respiratory metabolism in sink leaves it catalyzes a net glycolytic flux, as in potato tubers. However, tobacco seedlings are able to compensate for a large decrease in expression of PFP without loss of growth, or the ability to cope with suboptimal phosphate, nitrogen or temperature.

Contrasting Roles for Pyrophosphate Fructose-6-Phosphate Phosphotransferase During Aging of Tissue-Slices from Potato-Tubers and Carrot Storage Tissues

Abstract The role of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) during wounding-induced respiration in tissue slices from a starch-storing tissue (potato tubers) and a sucrose-storing tissue (carrot root) has been compared. (a) In both tissues, there was a rapid decrease of glycerate-3-phosphate, a gradual increase of triose-phosphate and fructose-1,6-bisphosphate, no change or an increase of hexose phosphates, and a rapid increase of fructose-2,6-bisphosphate. Inorganic pyrophosphate responded differently in the two tissues, decreasing in potato and increasing in carrot. (b) Comparison of key enzyme activities in extracts showed differences between the two tissues. Potato tubers contained high pyruvate kinase, relative to ATP-dependent phosphofructokinase, and also contained high PFP. Carrot roots contained comparable capacities of pyruvate kinase and ATP-dependent phosphofructokinase, and two-fold lower PFP. (c) It is argued that PFP probably has a glycolytic function in potato tubers, supplementing the rather low ATP-dependent phosphofructokinase activity. In carrot, ATP-dependent phosphofructokinase is relatively high, allowing PFP to operate in the reverse direction, and generate inorganic pyrophosphate which is needed for sucrose mobilisation.