Sucrose Synthase mRNA Research Articles

The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots

Sucrose utilisation in sink tissues depend on its cleavage and is mediated by two different classes of enzymes, invertase and sucrose synthase, which determine the mechanism of phloem unloading. Cloning of two extracellular (BIN35 and BIN46) and one vacuolar invertase (BIN44) provided the basis for a detailed molecular analysis of the relative contribution of the sucrose cleaving enzymes to the sink metabolism of sugar beets ( Beta vulgaris) during development. The determination of the steady state levels of mRNAs has been complemented by the analysis of the corresponding enzyme activities. The present study demonstrates an inverse regulation of extracellular invertase and sucrose synthase during tap root development indicating a transition between functional unloading pathways. Extracellular cleavage by invertase is the dominating mechanism to supply hexoses via an apoplasmic pathway at early stages of storage root development. Only at later stages sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength of the tap root via symplasmic phloem unloading. Whereas mRNAs for both extracellular invertase BIN35 and sucrose synthase were shown to be induced by mechanical wounding of mature leaves of adult plants, only sucrose synthase mRNA was metabolically induced by glucose in this source organ supporting the metabolic flexibility of this species.

Apoplastic Invertase and Its Enhanced Expression and Post-translation Control during Fruit Maturation and Ripening

An invertase gene was isolated and its mRNA activity and protein levels were determined during papaya (Carica papaya L.) fruit development. A complete invertase cDNA (AF420223) and a partial sucrose synthase cDNA (AF420224) were isolated from papaya fruit cDNA libraries. The invertase cDNA encoded a predicted polypeptide of 582 residues (MW 65,537 Da), and was 68% and 45% identical with carrot apoplastic and vacuolar invertases, respectively. Key amino acids indicative of an apoplastic invertase were conserved. A full-length gene corresponding to the putative apoplastic invertase cDNA was isolated and was organized into seven exons and six introns. Exon 2 (9 bp long) encoded part of a highly conserved region (NDPNG/A). Invertase mRNA and activity levels increased during fruit maturation and sugar accumulation just before ripening. In contrast, sucrose synthase mRNA levels were high during early fruit growth and low during the fruit sugar accumulation stage. A 73-kDa cell wall extractable protein that cross-reacted with carrot apoplastic invertase antisera substantially increased during papaya fruit maturation and declined in full ripe fruit. The increase in invertase protein levels occurred 2 to 4 weeks before maturity and was markedly higher than the overall increase in enzyme activity at this stage. Subsequently, the increase in enzyme activity was higher than the increase in protein levels between 2 weeks before maturity and fully ripe. The results suggested that mRNA level and invertase activity were related to maturity. The data suggested that the invertase was apoplastic, and that post-translational control of enzyme activity occurred, in which a significant accumulation of invertase occurred before the peak of enzymes activity.

Short-term metabolic responses of soybean root nodules to nitrate.

Soybean (Glycine max L. Merr.) plants exposed to 10 mM KNO(3) for a 4 d period were used to test the correlation between nitrogenase activity, gene expression and sucrose metabolism. Nitrate caused the down-regulation of sucrose synthase (SS) transcripts within 1 d, although a decline in nodule SS activity and an increase in nodule sucrose content only occurred after 3-4 d. In a second experiment, plants were exposed to (15)N-labelled nitrate for 48 h to determine the time period during which nitrate was taken up, and to relate this to the decline in apparent nitrogenase activity (H(2) production in air) and the reduction in SS gene transcript levels. The peak of nitrate uptake appeared to be between 8 h and 14 h whilst apparent nitrogenase activity began to decline at about 17.5 h. The SS mRNA signal declined markedly between 14 h and 24 h. The correlative association of these factors is clear. However, SS activity per se does not appear to be related to the initial decline in apparent nitrogenase activity as a result of nitrate uptake. These findings, therefore, do not support the hypothesis that the regulation of nodule function is mediated by the regulation of SS activity.

Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation.

In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was confirmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as filial tissues. In a series of in situ hybridizations we analysed the cell type-specific expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5-6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.

Stress-Induced Declines in Soybean N2 Fixation Are Related to Nodule Sucrose Synthase Activity.

Soybean (Glycine max L.) plants were subjected to a number of treatments (drought, 10 mM nitrate, 150 mM NaCl, shoot meristem removal, and removal of approximately 50% of the nodules) to test the hypothesis that metabolic responses contribute to the regulation of N2 fixation. Nitrogenase activity was correlated with the activity of nodule sucrose synthase (SS), but not with that of glutamine oxoglutarate amino transferase. Leghemoglobin levels and other enzyme activities were not significantly or consistently affected by the treatments. SS mRNA was greatly reduced in nodules of drought-, salt-, and nitrate-treated plants; however, this was not correlated with changes in soluble carbohydrate, starch, amino acids, or ureides. Leghemoglobin mRNA was only slightly affected by the treatments. The time course of drought stress showed a decline in the SS transcript level by 1 d, but levels of leghemoglobin, glutamine synthetase, and ascorbate peroxidase mRNA were not markedly affected by 4 d. SS activity at 4 d was reduced by 46%. We propose that N2 fixation in soybean nodules is mediated by both the oxygen-diffusion barrier and the potential to metabolize sucrose via SS. The response to environmental perturbation may involve down-regulation of the nodule SS gene.

Changes in levels of mRNAs for cell wall-related enzymes in growing cotton fiber cells.

mRNAs for cell wall-related enzymes in developing cotton fiber cells were measured by reverse transcription-PCR analysis. Both endo-1,4-beta-glucanase and expansin mRNA levels were high during cell elongation but decreased when cell elongation ceased, and xyloglucan decreased. The endo-1,3-beta-glucanase mRNA level was very low in the elongating cells but increased gradually at the onset of secondary wall synthesis, accompanying the massive deposition of cellulose. Endoxyloglucan transferase and sucrose synthase mRNA levels were constant during all stages of growth.

Sucrose Synthase Localization during Initiation of Seed Development and Trichome Differentiation in Cotton Ovules.

Sucrose synthase in cotton (Gossypium hirsutum L.) ovules was immunolocalized to clarify the relationship between this enzyme and (a) sucrose import/utilization during initiation of seed development, (b) trichome differentiation, and (c) cell-wall biosynthesis in these rapidly elongating "fibers." Analyses focused on the period immediately before and after trichome initiation (at pollination). Internal tissues most heavily immunolabeled were the developing nucellus, adjacent integument (inner surface), and the vascular region. Little sucrose synthase was associated with the outermost epidermis on the day preceding pollination. However, 1 d later, immunolabel appeared specifically in those epidermal cells at the earliest visible phase of trichome differentiation. The day following pollination, these cells had elongated 3- to 5-fold and showed a further enhancement of sucrose synthase immunolabel. Levels of sucrose synthase mRNA also increased during this period, regardless of whether pollination per se had occurred. Timing of onset for the cell-specific localization of sucrose synthase in young seeds and trichome initials indicates a close association between this enzyme and sucrose import at a cellular level, as well as a potentially integral role in cell-wall biosynthesis.

Inhibitors of Protein Phosphatases 1 and 2A Block the Sugar-Inducible Gene Expression in Plants.

Genes coding for two major proteins of the tuberous root of sweet potato (Ipomoea batatas), namely, sporamin and [beta]-amylase, are inducible in leaves and petioles when they are supplied with high concentrations of sucrose or other metabolizable sugars, such as glucose and fructose, and the accumulation of a large amount of starch accompanies this induction. Three inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), namely, okadaic acid, microcystin-LR, and calyculin A, strongly inhibited the sucrose-inducible accumulation of mRNAs for sporamin, [beta]-amylase, and the small subunit of ADP-glucose pyrophosphorylase in petioles. However, these inhibitors did not have any major effect on the steady-state levels of mRNAs for catalase and glyceraldehyde-3-phosphate dehydrogenase, and the sucrose-inducible increase in the level of sucrose synthase mRNA was enhanced by okadaic acid. Inhibitors of PP1 and PP2A also inhibited sucrose-inducible expression of a fusion gene, consisting of the promoter of the sweet potato gene for [beta]-amylase and the coding sequence for [beta]-glucuronidase (GUS), in leaves of transgenic tobacco (Nicotiana tabacum). The inhibition was not due to inhibition of uptake and cleavage of sucrose, since okadaic acid also inhibited induction of the fusion gene by glucose or fructose. Addition of okadaic acid to leaves that had been treated with sucrose for 6 h inhibited further increases in GUS activity. These results suggest that the continuous dephosphorylation of proteins is required in the transduction of carbohydrate metabolic signals to the transcriptional activation of at least some sugar-inducible genes in plant.

Involvement of intracellular calcium in anaerobic gene expression and survival of maize seedlings.

Ca-mediated processes are known to be involved in transducing many developmental, hormonal, and environmental cues in plant cells. In this study, the role of Ca in the perception of anoxic stress signals by maize (Zea mays L. cv B73) roots was assessed by studying the effect of various Ca antagonists on the induction of alcohol dehydrogenase (ADH) and sucrose synthase mRNA as well as ADH activity under anoxia. The effect of these compounds on the poststress recovery of the seedlings was also monitored. Ruthenium red (RR), an inhibitor of organellar Ca fluxes, repressed the anoxic activation of the alcohol dehydrogenase1 and shrunken1 genes as measured by their transcript levels as well as ADH activity. Furthermore, RR-treated seedlings could not recover even after only 2 h of flooding, in contrast to untreated B73 seedlings that survived 72 h of submergence. Ca, when supplied along with RR, allowed normal anoxic gene expression and also prevented the RR-imposed death of the seedlings from short-term anoxia. Ca (45Ca) fluxes were measured in maize roots to elucidate the mode of action of RR. RR abolished anoxia-stimulated 45Ca influx into maize roots but did not affect aerobic Ca2+ uptake, unlike a few other antagonists that blocked both the aerobic and anoxic fluxes. However, Ca uptake across the plasma membrane was not necessary for the adaptive response to anoxia, since chelation of extracellular Ca by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid did not affect the induction of ADH activity or poststress survival of flooded seedlings. The data suggest that RR may act on one of the intracellular stores of Ca and the Ca mobilized from this source is a physiological transducer of anoxic stress signals in maize roots.

Temporal and Spatial Expression Pattern of Sucrose Synthase during Tomato Fruit Development.

Sucrose synthase is proposed to play an important role in the early stages of tomato fruit (Lycopersicon esculentum Mill.) growth. In this work, the temporal and spatial expression patterns of sucrose synthase during tomato fruit development were investigated. Fruit contained the majority of the sucrose synthase protein and mRNA relative to other organs. Only trace levels of sucrose synthase protein and mRNA were detected in the stem, petiole, and roots. Sucrose synthase mRNA was detected in pistils prior to anthesis, reached peak levels in fruit 5 to 7 d after anthesis (DAA), and was not detectable after 35 DAA. Sucrose synthase protein levels reached a maximum at 20 to 25 DAA and then declined to nondetectable levels after 45 DAA. The lack of coordination between protein and mRNA levels suggests that sucrose synthase expression may be controlled at the levels of both transcription and translation. Sucrose synthase mRNA was differentially localized in the fruit, being most abundant in the mesocarp cells adjacent to the placenta, the columella, and the cells surrounding the vascular bundle. Except around the vascular tissue, the localization of sucrose synthase mRNA positively correlates with starch granule accumulation at the cellular level.

Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level

The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere. Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level. The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5-6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed. Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44.

Anaerobic Stress Induces the Transcription and Translation of Sucrose Synthase in Rice

Sucrose synthase activity increased in 2-day-old rice (Oryza sativa) seedlings submitted to anaerobic stress. Likewise, both denaturing and native Western blot analysis detected a rise in the cellular concentration of sucrose synthase protein. Significantly higher steady-state levels of sucrose synthase mRNA, as determined by Northern blots and by the ability of total RNA to direct in vitro synthesis of sucrose synthase, were also induced by anaerobic treatment. Analysis of run-on transcripts showed increased transcription of sucrose synthase genes as early as 60 minutes after initiation of anaerobic stress. Together, these results indicate that sucrose synthase is a typical anaerobic protein in rice.

The steady-state level of potato sucrose synthase mRN A is dependent on wounding, anaerobiosis and sucrose concentration

The steady-state level of potato sucrose synthase (SSase) mRNA was investigated in different plant organs and in response to certain stimuli. SSase mRNA is mainly present in developing tubers, but its presence is not restricted to this organ and the transcript is found at detectable levels in all the tissues analysed, except leaves. Wounding results in a decrease in SSase mRNA, but this can be overcome by incubation under anaerobic conditions. In leaves and petioles, an increase in sucrose concentration leads to an increase in SSase mRNA.

Anaerobiosis Induces Transcription but Not Translation of Sucrose Synthase in Maize

This report examines the effect of anaerobic stress on the expression of sucrose synthase in maize (Zea mays L.). Following 24 hours of anaerobic treatment, alcohol dehydrogenase displayed the classical characteristics of induction: increased mRNA and protein levels. However, there was no detectable increase in sucrose synthase specific proteins by either native or denaturing Western blot analysis nor was there an increase in sucrose synthase activity. Anaerobic treatment did induce significantly higher steady state levels of sucrose synthase mRNA. Even though previous work has implicated sucrose synthase as an anaerobically induced protein, the data in this report suggest that sucrose synthase is not inducible at the protein level by anaerobic treatment.

Molecular cloning and sequencing of sucrose synthase cDNA from potato ( Solanum tuberosum L.): preliminary characterization of sucrose synthase mRNA distribution

A cDNA library constructed from poly(A) + RN A from potato tuber in λgt 11 was screened for sucrose synthase using a maize sucrose synthase cDNA probe. The longest 2.7-kb insert was sequenced. An ATG located within the sequence CTGCAATGG starts an open reading frame of 805 codons. The nucleotide sequence, when compared to the maize sucrose synthase cDNA sequence, exhibits about 70% identity and the deduced amino acid sequence about 75%. Three amino acid regions are about 90% homologous and two of them could be important for the protein function. Expression studies show that transcription of the potato sucrose synthase gene is at least ten fold higher in tubers compared to photosynthetically active tissues.

Translation of Zea mays endosperm sucrose-synthase mRNA in vitro.

mRNA of 23-day-old maize endosperm was translated both in wheat germ extracts and rabbit reticulocyte lysates. A protein with an apparent molecular weight of 88,000 comigrates in dodecylsulfate/polyacrylamide electrophoresis with sucrose synthase. This protein is precipitated with an antiserum against sucrose synthase and shows the same protease digestion pattern as the enzyme. It is not synthesized with mRNA extracted from sh/sh mutant kernels lacking sucrose synthase. By these criteria, the protein is the translation product in vitro of sucrose synthase mRNA. The separation of mRNA in methylmercury-hydroxide--agarose gels and subsequent translation indicates a length of sucrose synthase mRNA of 2800 nucleotides which is compatible with the coding length necessary for a protein with a molecular weight of 88,000 plus untranslated sequences.