Horacio G. Pontis

New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking.

The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach and Arabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized the ArabidopsisAt-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. The At-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage in Arabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants.

Differential expression of alkaline and neutral invertases in response to environmental stresses: characterization of an alkaline isoform as a stress-response enzyme in wheat leaves

It is well accepted that sucrose (Suc) metabolism is involved in responses to environmental stresses in many plant species. In the present study we showed that alkaline invertase (A-Inv) expression is up-regulated in wheat leaves after an osmotic stress or a low-temperature treatment. We demonstrated that the increase of total alkaline/neutral Inv activity in wheat leaves after a stress could be due to the induction of an A-Inv isoform. Also, we identified and functionally characterized the first wheat cDNA sequence that codes for an A-Inv. The wheat leaf full-length sequence encoded a protein 70% similar to a neutral Inv of Lolium temulentum; however, after functional characterization, it resulted to encode a protein that hydrolyzed Suc to hexoses with an optimum pH of 8, and, consequently, the encoding sequence was named Ta-A-Inv. By RT-PCR assays we demonstrated that Ta-A-Inv expression is induced in response to osmotic and cold stress in mature primary wheat leaves. We propose that Ta-A-Inv activity could play an important role associated with a more efficient cytosolic Suc hydrolysis during environmental stresses.

STUDIES ON SUCROSE PHOSPHATE SYNTHETASE The inhibitory action of sucrose

Plant biochemists are confronted with two path- ways leading to sucrose, both of which involve UDP- glucose as glucosyl donor. Sucrose synthetase [l] (UDP-glucose: D-fructose-2glucosyltransferase, EC 2.4.2.13) and sucrose phosphate synthetase [2] (UDP-glucose: D-fructose-6-phosphate-Zglucosyl- transferase, EC 2.4.2.14) with its associate sucrose phosphate phosphatase [3,4] (sucrose8-phosphate phosphohydrolase, EC 3.1.3 .OO) catalyze the follow- ing reactions : UDP-glucose t fructose ti UDP t sucrose UDP-glucose + fructose-6-P 2 UDP t sucrose-6-P sucrose-6-P 4 sucrose + Pi The existence of these two separate mechanisms for the synthesis of sucrose raises the question of their respective roles in vivo [ 51. Present views tend to stress that the physiological role of sucrose synthetase would be sucrose cleavage, while the role of sucrose phosphate synthetase coupled to sucrose phosphate phosphatase would correspondingly be that of sucrose synthesis [6]. Thus, sucrose synthesis would be the * It

A procedure for the assay of sucrose synthetase and sucrose phosphate synthetase in plant homogenates

The determination of these two enzymic activities is commonly based on the destruction of remaining fructose or fructose-6-P by treatment with borohydride or alkali (1,2,4). These methods are quite suitable for kinetic determinations, but they are not adequate for testing the enzymes in crude homogenates from various plant tissues. In this case, methods which measure the formation of [14C]sucrose from UDP-[14C]glu cose have been used. Radioactive sucrose is separated from the labeled substrate by paper electrophoresis (5) or by paper chromatography (6). Similarly, the labeled sucrose phosphate in the reaction catalyzed by sucrose phosphate synthetase is separated from the substrate by paper chromatography after submitting the ester to the action of alkaline phosphatase (7). In the latter case a similar technique is applied if radioactive fructose-6-P is used instead of UDP-glucose (7). Thus, these procedures, even if they give reproducible results, are

Purification and properties of the soluble acid invertase from Oryza sativa

Abstract An invertase (β-fructofuranosidase, EC 3.2.1.26) from aerial parts of 17-day-old Oryza sativa plants has been purified to homogeneity by gel filtration on Sephadex G-150 and adsorption on brushite. The invertase is a dimeric glycoprotein (Mr 98 000) composed of two identical subunits (Mr 46 000). The pI is ca 6 and the activation energy is 8250 cal mol−1 over 30° and 24 000 cal mol−1 below this temperature. The enzyme shows a high specificity for sucrose (Km 6.6 × 10−3M) and its activity is modulated by fructose. Glucose is a classical non-competitive inhibitor and fructose produces a complex non-linear competitive inhibition. Proteins are activators of the invertase, but they do not suppress the inhibitory action of the reaction products. The in vitro spontaneous complex formation among invertases and proteins in higher plants suggests that invertases are functioning complexed with some protein in vivo.

Observations on the de Novo synthesis of fructosans in vivo.

Abstract Expiants of mature dormant tubers of Helianthus tuberosus were grown in the absence of carbon source with different auxins. After transfer to a glucose medium, the explants were able to initiate a de novo synthesis of fructosans. This synthesis was affected by auxins, kinetin, and gibberellic acid. Dinitrophenol (1 × 10 −4 m ) produced an inhibition of synthesis of between 65 and 80% when added to the medium 12 hours after the carbohydrate. When protein synthesis was inhibited by chloramphenicol the synthesis of fructosans was also blocked.

Studies on the sucrose phosphate synthetase kinetic mechanism

Abstract The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.

Transport of 1-kestose across the tonoplast of Jerusalem artichoke tubers

The capacity for 1-kestose uptake into the vacuole of fructan storing Jerusalem artichoke tubers was investigated. 1-kestose serves both as building block for fructan initiation and as a fructose donor for chain elongation. Tonoplast vesicles were isolated from actively storing tubers, and their vesicles were capable of transporting sucrose in a manner indicative of a sucrose/H(+) antiport. Under similar conditions, 1-kestose was not taken up by vesicles energized by either a pH jump or in the presence of ATP. When added together at 2 mM, sucrose uptake was not affected by the presence of 1-kestose. The data argues against the possible synthesis of 1-kestose in the cytosol and subsequent transport to the vacuole. The data also presents definite evidence for the existence a mechanism for sucrose accumulation in fructan storing vacuoles.