Kinetic model of sucrose accumulation in maturing sugarcane culm tissue

Biochemically, it is not completely understood why or how commercial varieties of sugarcane (Saccharum officinarum) are able to accumulate sucrose in high concentrations. Such concentrations are obtained despite the presence of sucrose synthesis/breakdown cycles (futile cycling) in the culm of the storage parenchyma. Given the complexity of the process, kinetic modelling may help to elucidate the factors governing sucrose accumulation or direct the design of experimental optimisation strategies. This paper describes the extension of an existing model of sucrose accumulation (Rohwer, J.M., Botha, F.C., 2001. Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochem. J. 358, 437-445) to account for isoforms of sucrose synthase and fructokinase, carbon partitioning towards fibre formation, and the glycolytic enzymes phosphofructokinase (PFK), pyrophosphate-dependent PFK and aldolase. Moreover, by including data on the maximal activity of the enzymes as measured in different internodes, a growth model was constructed that describes the metabolic behaviour as sugarcane parenchymal tissue matures from internodes 3-10. While there was some discrepancy between modelled and experimentally determined steady-state sucrose concentrations in the cytoplasm, steady-state fluxes showed a better fit. The model supports a hypothesis of vacuolar sucrose accumulation against a concentration gradient. A detailed metabolic control analysis of sucrose synthase showed that each isoform has a unique control profile. Fructose uptake by the cell and sucrose uptake by the vacuole had a negative control on the futile cycling of sucrose and a positive control on sucrose accumulation, while the control profile for neutral invertase was reversed. When the activities of these three enzymes were changed from their reference values, the effects on futile cycling and sucrose accumulation were amplified. The model can be run online at the JWS Online database (http://jjj.biochem.sun.ac.za/database/uys).     

Sugar transport in immature internodal tissue of sugarcane: I. Mechanism and kinetics of accumulation

Transmembrane sugar transport into immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L.) is a metabolically regulated process as evidenced by its sensitivity to pH, temperature, anaerobiosis, and metabolic inhibitors. All sugars studied—glucose, fructose, galactose, sorbose, glucose 6-phosphate, 3-O-methylglucose, and 2-deoxy-d-glucose—were apparently transported via the same carrier sites since they competed with each other for uptake. External concentrations of these sugars at one-half V_max were in the range of 3.9 to 8.4 nm. Preliminary data indicated that phosphorylation may be closely associated with glucose transport. The dominant intracellular sugar after 4-hours incubation was sucrose when glucose, glucose-6-P, or fructose was the exogenously supplied sugar; but when galactose was supplied, only 28% of intracellular radioactivity was in sucrose. Sorbose, 3-O-methylglucose, and 2-deoxy-d-glucose were not metabolized. Thus, by using these analogs, transport could be studied independently of subsequent metabolism, effectively eliminating a complicating factor in previous studies.     

Vascular changes in maturing granulation tissue

Granulation tissue was examined in rats on the 20th and 40th day after wounding of the back. It was shown that by both the 20th and the 40th day the vessels with impaired typical structure could be revealed together with the normal ones in the granulation tissue. Specific rearrangement was observed in the structure of these vessels, consisting in the impairment of their wall integrity, separation of the constituent cells and their free position among the other cells and fibrous structures. This process is especially marked by the 40th day. 3H-thymidine was actively incorporated by some fibroblasts and rather often by the cells of both normal and degenerating vessels. The authors suggest the existence of an earlier unknown phenomenon of transformation of capillary vessels, common for both normal dermal and reparative processes after injury. The essence of the phenomenon consists in the fact that small dermal vessels get permanently disintegrated and are included into the composition of cellular elements of the interstitial tissue, and form again, providing for the physiological regeneration of dermal cells and fibrous structures.     

A novel spermatogenesis-related factor-1 gene expressed in maturing rat testis.

A rat gene with testis-specific expression coinciding with spermatogenesis was cloned by differential display. This spermatogenesis-related factor-1 (SRF-1) gene was not expressed in other organs. Testicular expression was detected from 5 weeks of age and increased up to 15 weeks; this level of expression was maintained for 63 weeks. The 750-bp cloned gene was coded for an open reading frame of 202 amino acids. According to in situ hybridization at 7 weeks, this gene was expressed mainly in spermatocyte. The gene product may function as a molecular motor in meiosis, as the deduced amino acid sequence showed partial homology with kinesin-related proteins. The action of this gene and its product with respect to division of reproductive cells requires further investigation.     

Sugar transformations associated with hexose transport in immature internodal tissue of sugar cane

After a 15 sec incubation in d-glucose-¹⁴C(U), 53–70% of the intracellular radioactivity in immature internodal tissue of sugarcane was in glucose-6-phosphate, and the remainder was in free glucose. Two unmetabolized glucose analogs, 2-deoxy-d-glucosce and 3-O-methyl-d-glucose, were transported at rates comparable to glucose but neither of these analogs was phosphorylated. Doubly-labeled d-glucose-1-¹⁴C-6-phosphate-³²P was dephosphorylated prior to deposition in the inner space, and ¹⁴C was transported into this tissue twice as rapidly as ³²P. It was also shown that ³²P in exogenously supplied glucose-6-³²P was not the source of phosphate for the intracellular synthesis of glucose-6-P. Galactose transport was similar to that of glucose in that the first major product recovered intracellularly was a phosphorylated sugar, i.e. ¹⁴C-galactose-1-P, when the tissue was incubated in d-galactose-¹⁴C(U). Although fructose, glucose, and galactose competed for transport into this tissue, free fructose and glucose predominated in the tissue extract after a 15-sce incubation in d-fructose-¹⁴C(U). This contrasted sharply, with the products of ¹⁴C-glucose transport which were comprised of phosphorylated sugars after 15 sec.     

Compartmentation of solutes and water in developing sugarcane stalk tissue

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from −0.6 to −1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from −1.0 to approximately −2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.     

Genes expressed during sexual differentiation of Chlamydomonas reinhardtii

Four genes specifically expressed during gametogenesis of Chlamydomonas reinhardtii have been cloned and their expression patterns analyzed. mRNAs encoded by these gamete-specific genes (gas) were absent or present only at very low levels in vegetative cells and mature zygotes. In young zygotes 2 h after gamete fusion, the mRNAs of three gas genes still persisted. The gas mRNAs accumulated during gametic differentiation. The temporal patterns of accumulation of individual mRNAs differed; some started to increase early during gametogenesis, others accumulated in the late phase. The accumulation of one of the late mRNAs (gas28) was stricly light-dependent. To illustrate the utility of the genes cloned in the analysis of sexual differentiation in Chlamydomonas reinhardtii we show that in a gametogenesis-defective mutant, the expression of late genes is prevented while that of early genes is normal.     

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.     

Genes expressed in the amphioxus notochord revealed by EST analysis

The notochord cell of the cephalochordate amphioxus adult is unique due to the occurrence of myofilaments in the cytoplasm. The present EST (expressed sequence tag) analysis targeted mRNAs of the amphioxus notochord to determine genes that are expressed there. Notochord cells were isolated from Branchiostoma belcheri adults, from which a cDNA library was constructed. Analysis of a set of 257 ESTs (both 5' and 3' ends) showed that about 11% of the cDNAs are related to muscle genes, while 9% of them are genes for extracellular matrix proteins associated with formation of the notochordal sheath. The muscle-related genes included actin, tropomyosin, troponin I, myosin regulatory light chain, myosin light chain kinase, myosin heavy chain, calmodulin, calponin, calcium vector protein, creatine kinase, muscle LIM protein, and SH3-binding glutamate-rich protein, suggesting that vertebrate skeletal and smooth muscle-type genes are simultaneously expressed in the amphioxus notochord. Nucleotide sequences of cDNAs for actin, tropomyosin, troponin I, and a few others were completely determined to substantiate the conclusions. The chordate muscle-type actin is distinguishable from the cytoplasmic-type actin by the usage of amino acid residues at 20 diagnostic positions. Interestingly, analysis of the usage of amino acid residues at these positions showed that the "amphioxus notochord actin" is a unique intermediate between muscle-type and cytoplasmic-type actins. These results strongly suggest that the notochord of adult amphioxus is a mechanical swimming organ and its role is quite different from the role of the vertebrate embryonic notochord, which functions as a source of signals required for body plan formation.     

Genes specifically expressed in sexually differentiated female spheroids of Volvox carteri

Volvox carteri is a multicellular green alga with only two cell types, somatic cells and reproductive cells. Phylogenetic analysis suggests that this organism has evolved from a Chlamydomonas-like unicellular ancestor along with multicellularity, cellular differentiation, and a change in the mode of sexual reproduction from isogamy to oogamy. To examine the mechanism of sexual differentiation and the evolution of oogamy, we isolated 6 different cDNA sequences specifically expressed in sexually differentiated female spheroids. The genes for the cDNAs were designated SEF1 to SEF6. The time course of accumulation of each mRNA was shown to be distinct. The expression of some of these genes was not significantly affected when the sexual inducer was removed after the induction of sexual development. Sequence analysis indicates that SEF5 and SEF6 encode pherophorin-related proteins. Of these, SEF5 has the unique structural feature of a polyproline stretch in the C-terminal domain in addition to the one found in the central region.     

Genes specifically expressed at growth arrest of mammalian cells

A subtraction cDNA library enriched for RNA sequences preferentially expressed in growth-arrested cells was prepared. Six cDNA clones were identified, varying in abundance from 2% to 0.0002% of the library and in size from 0.8 to 10 kb. The corresponding mRNAs are downregulated with different kinetics upon induction of growth by serum. The kinetics of induction after serum starvation and density-dependent inhibition of two of these growth-arrest-specific (gas) genes were investigated in more detail. Two cell lines transformed by viral onc genes did not express the two gas genes. The full-length cDNA for one gene has been sequenced and the protein product preliminarily characterized by in vitro translation.     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.