Sucrose Phosphate Phosphatase Research Articles

Comparative Analysis of Sucrose-Regulatory Genes in High- and Low-Sucrose Sister Clones of Sugarcane.

Sugarcane is a significant primitive source of sugar and energy worldwide. The progress in enhancing the sugar content in sugarcane cultivars remains limited due to an insufficient understanding of specific genes related to sucrose production. The present investigation examined the enzyme activities, levels of reducing and non-reducing sugars, and transcript expression using RT-qPCR to assess the gene expression associated with sucrose metabolism in a high-sucrose sugarcane clone (GXB9) in comparison to a low-sucrose sister clone (B9). Sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), sucrose synthase (SuSy), cell wall invertase (CWI), soluble acid invertase (SAI), and neutral invertase (NI) are essential enzymes involved in sucrose metabolism in sugarcane. The activities of these enzymes were comparatively quantified and analyzed in immature and maturing internodes of the high- and low-sucrose clones. The results showed that the higher-sucrose-accumulating clone had greater sucrose concentrations than the low-sucrose-accumulating clone; however, maturing internodes had higher sucrose levels than immature internodes in both clones. Hexose concentrations were higher in immature internodes than in maturing internodes for both clones. The SPS and SPP enzymes activities were higher in the high-sucrose-storing clone than in the low-sucrose clone. SuSy activity was higher in the low-sucrose clone than in the high-sucrose clone; further, the degree of SuSy activity was higher in immature internodes than in maturing internodes for both clones. The SPS gene expression was considerably higher in mature internodes of the high-sucrose clones than the low-sucrose clone. Conversely, the SuSy gene exhibited up-regulated expression in the low-sucrose clone. The enhanced expression of SPS in the high-sucrose clone compared to the low-sucrose clone suggests that SPS plays a major role in the increased accumulation of sucrose. These findings provide the opportunity to improve sugarcane cultivars by regulating the activity of genes related to sucrose metabolism using transgenic techniques.

Identification of Solanum pimpinellifolium genome regions for increased resilience to nitrogen deficiency in cultivated tomato

High-quality crop production with minimal fertilizer inputs is a key goal for the agriculture of the future. Globally, tomato is one of the most important vegetable crops and its intensive production and breeding has been based on the application of large quantities of nitrogen (N) fertilizers. Therefore, the development of N use efficient (NUE) cultivars with low N inputs needs to be addressed. Some variability in plant growth, fruit quality and NUE traits among tomato (Solanum lycopersicum L.) varieties under low N supply has been reported, however, the relevance of wild relatives of tomato has not yet been assessed. In this study, we found that S. pimpinellifolium accession To-937 (SP) may be a suitable resource to increase NUE in tomato. We studied a set of 29 introgression lines (IL) from SP into the Moneymaker cultivar (MM) in different seasons to investigate the potential of SP introgressions to maintain the tomato plant performance during the growth cycle under low N input in greenhouse conditions. We identified specific regions in the SP genome, on chromosomes 1, 3 and 10, involved in the responses to N inputs of fruit production and fruit quality. Notably, the line SP_10-4 maintained vegetative biomass and fruit yield production under limiting N supply. The introgressed region contained putative candidate genes as sucrose phosphate phosphatase (SPP), invertases (INV) and glutamine synthase 1 (GS1) genes, implicated in C and N metabolism. Genomic and expression analyses revealed differences in coding and non-coding sequences as well as in mRNA levels in SP_10-4, suggesting that these genes might well contribute to the reported biomass responses to N. Additionally, line SP_1-4 showed stable fruit amino acid contents under both sufficient and limiting N supplies, indicating that assimilated N partitioning to the fruit is maintained in response to N. Altogether, our results confirmed the suitability of SP as a source of NUE related traits and the interest in the studied ILs for developing new tomato cultivars with improved NUE under sustainable fertilization conditions.

Pericarp starch metabolism is associated with caryopsis development and endosperm starch accumulation in common wheat

The wheat pericarp is the main component of the caryopsis at the early development stage and ultimately converts into a tissue that covers the mature caryopsis. A large number of starch granules are accumulated in the pericarp, but the production of and the role of starch granules in caryopsis development remain- elusive. In the present study, the relationship between accumulated starch granules and starch metabolism-related genes in wheat pericarp was investigated using paraffin section observations, expression analysis, and mutant analysis. Starch synthesis is initiated before anthesis and is dependent on a sucrose uptake and conversion system similar to that in the endosperm. TaPTST2 is required to initiate the production of pericarp starch granules. Pericarp starch granules gradually disappeared at the filling stage with high expression levels of genes encoding β-amylase, sucrose-phosphate synthase, and sucrose-phosphate phosphatase. As a maternal tissue adjacent to the endosperm and embryo, the pericarp plays a temporary reservoir for excess nutrients delivered into the caryopsis during the early development stage and exported at the filling stage. The pericarp contributes to the development of the endosperm and embryo as well as the accumulation of endosperm starch. The metabolism of pericarp starch may affect the weight of the wheat caryopsis.

Coffee's carbohydrates. A critical review of scientific literature

Only two species have gained economic importance in coffee production: Coffea arabica L. (Arabica coffee) & Coffea canephora Pierre ex A. Froehner var. Robusta, with 65 and 35% of world production attributed to C. arabica http://wsx5customurl.comL. & C. canephora P. respectively. In general, it is estimated that 6 mt of fresh and ripe fruits produce approximately 1 mt of raw and dry grains. The grain endosperm is mainly composed of cellulose, hemicelluloses, proteins, minerals and lipids, but starch and tannins are absent. However, the seed's chemical composition of C. arabica and C. canephora, before roasting, differs concerning their primary and secondary metabolites content, which serve as precursors for the synthesis of volatile compounds during the roasting process. For this reason, there are marked organoleptic differences between both species' roasted and ground grain. However, the evidence suggests that such differences can also be attributed to other factors since coffees grown in cool, highland areas generally have better sensory attributes than their counterparts grown in hot, lowland areas. It has been speculated that environmental conditions in cool, highland areas induce the slow accumulation of primary and secondary metabolites during the endosperm development resulting in sensorial differences after roasting. This essay focuses on the study of coffee beans' carbohydrates (primary metabolites) before and after roasting, their influence on cup quality, biosynthesis and differences linked to the involved species, their metabolism, solubility and extraction, as well as a discussion on the analytical techniques used for its determination. Keywords: sucrose synthase, sucrose phosphate phosphatase, sucrose phosphate synthase, aploplasm, cytoplasm, Manan synthase, Galactosyl transferase.

Transcription Profiles Reveal Age-Dependent Variations of Photosynthetic Properties and Sugar Metabolism in Grape Leaves (Vitis vinifera L.).

Leaves, considered as the ‘source’ organs, depend on the development stages because of the age-dependent photosynthesis and assimilation of leaves. However, the molecular mechanisms of age-dependent limitations on the function of leaves are seldom reported. In the present study, the photosynthesis-related characteristics and photoassimilates were investigated in grape leaves at six different age groups (Ll to L6) at micro-morphological, biochemical, and molecular levels. These results showed lower expression levels of genes associated with stomatal development, and chl biosynthesis resulted in fewer stomata and lowered chlorophyll a/b contents in L1 when compared to L3 and L5. The DEGs between L5 and L3/L1 were largely distributed at stomatal movement, carbon fixation, and sucrose and starch metabolism pathways, such as STOMATAL ANION CHANNEL PROTEIN 1 (SLAC1), FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE (FBA1), SUCROSE-PHOSPHATE SYNTHASE (SPP1), and SUCROSE-PHOSPHATE PHOSPHATASE (SPS2, 4). These genes could be major candidate genes leading to increased photosynthesis capacity and sugar content in L5. The accumulation of starch grains in the chloroplast and palisade tissue of L5 and higher transcription levels of genes related to starch biosynthesis in L5 further supported the high ability of L5 to produce photoassimilates. Hence, our results provide insights for understanding different photosynthetic functions in age-dependent leaves in grape plants at the molecular level.

Comparison of the root, leaf and internode transcriptomes in sugarcane (Saccharum spp. hybrids)

• The organs of sugarcane use carbon in different ways, leading to differing ratios of insoluble and soluble compounds • Transcription aligned closely with differences in the biomass makeup of insoluble and soluble components. • Large transcriptional differences were observed among organs, and smaller differences we observed according to maturity. • This study may support efforts to develop sugarcane with biomass composition better suited to various end uses. The major carbon source and sink organs (roots, leaves and internodes) of sugarcane ( Saccharum spp. hybrids) have different roles and consequently different apportioning of carbon into the major components of these organs. This study performed RNA-seq on two leaves, three internodes (of differing maturity) and roots in two similar commercial sugarcane genotypes to determine the patterns of gene expression associated with differences in carbon partitioning. A total of 18 RNA-seq libraries (in triplicate) were generated per genotype, thus yielding ∼0.6 billion high quality reads, with an average length of 125 bp, covering 90% of the long-read SUGIT transcriptome. RNA-seq, differential gene expression analysis, annotation and pathway analysis were performed. Transcript expression in active source organs (1st and 5th visible dewlap leaves) displayed higher expression in photosynthetic transcripts and some protein and respiratory transcripts, and low expression of most transcripts associated with cell-wall synthesis. Additionally, the transcription of some sucrose synthetic transcripts (sucrose phosphate synthase and sucrose phosphate phosphatase) was significantly higher than that in all other organs. Storage sinks (middle and bottom internodes) displayed lower expression of transcripts associated with UDP-glucose synthesis from sucrose and UDP-glucose conversion into cell-wall components than did meristematic sinks (immature internodes and root). Both meristematic sinks displayed lower transcript expression related to photosynthesis, and far higher expression of protein, cell-wall and respiratory-related transcripts, particularly in relation to source organs. Interestingly, transcription was higher for transcripts associated with lignin and hemicellulose synthesis in the roots but not immature internodes. Transcription closely aligned with differences in the biomass makeup of insoluble and soluble components in sugarcane organs. Similar transcriptional profiles observed across internodes of differing maturity, i.e., between immature and mature internodes, suggested that transcriptional changes in select sets of genes influence changes in the direction of carbon partitioning during maturation. This finding contrasts with the large transcriptional differences observed among major organ types. This knowledge may support efforts to develop sugarcane with biomass composition better suited to various end uses.

Proteome changes and associated physiological roles in chickpea (Cicer arietinum) tolerance to heat stress under field conditions.

Interrogative proteome analyses are used to identify and quantify the expression of proteins involved in heat tolerance and to identify associated physiological processes in heat-stressed plants. The objectives of the study were to identify and quantify the expression of proteins involved in heat tolerance and to identify associated physiological processes in chickpea (Cicer arietinum L.) heat-tolerant (Acc#7) and sensitive genotype (Acc#8) from a field study. Proteomic and gene ontological analyses showed an upregulation in proteins related to protein synthesis, intracellular traffic, defence and transport in the heat-tolerant genotype compared to the susceptible one at the warmer site. Results from KEGG analyses indicate the involvement of probable sucrose-phosphate synthase (EC 2.4.1.14) and sucrose-phosphate phosphatase (EC 3.1.3.24) proteins, that were upregulated in the heat-tolerant genotype at the warmer site, in the starch and sucrose pathway. The presence of these differentially regulated proteins including HSP70, ribulose bisphosphate carboxylase/oxygenase activase, plastocyanin and protoporphyrinogen oxidase suggests their potential role in heat tolerance, at flowering growth stage, in field-grown chickpea. This observation supports unaltered physiological and biochemical performance of the heat-tolerant genotypes (Acc#7) relative to the susceptible genotype (Acc#8) in related studies (Makonya et al. 2019). Characterisation of the candidate proteins identified in the current study as well as their specific roles in the tolerance to heat stress in chickpea are integral to further crop improvement initiatives.

Key Regulators of Sucrose Metabolism Identified through Comprehensive Comparative Transcriptome Analysis in Peanuts.

Sucrose content is a crucial indicator of quality and flavor in peanut seed, and there is a lack of clarity on the molecular basis of sucrose metabolism in peanut seed. In this context, we performed a comprehensive comparative transcriptome study on the samples collected at seven seed development stages between a high-sucrose content variety (ICG 12625) and a low-sucrose content variety (Zhonghua 10). The transcriptome analysis identified a total of 8334 genes exhibiting significantly different abundances between the high- and low-sucrose varieties. We identified 28 differentially expressed genes (DEGs) involved in sucrose metabolism in peanut and 12 of these encoded sugars will eventually be exported transporters (SWEETs). The remaining 16 genes encoded enzymes, such as cell wall invertase (CWIN), vacuolar invertase (VIN), cytoplasmic invertase (CIN), cytosolic fructose-bisphosphate aldolase (FBA), cytosolic fructose-1,6-bisphosphate phosphatase (FBP), sucrose synthase (SUS), cytosolic phosphoglucose isomerase (PGI), hexokinase (HK), and sucrose-phosphate phosphatase (SPP). The weighted gene co-expression network analysis (WGCNA) identified seven genes encoding key enzymes (CIN, FBA, FBP, HK, and SPP), three SWEET genes, and 90 transcription factors (TFs) showing a high correlation with sucrose content. Furthermore, upon validation, six of these genes were successfully verified as exhibiting higher expression in high-sucrose recombinant inbred lines (RILs). Our study suggested the key roles of the high expression of SWEETs and enzymes in sucrose synthesis making the genotype ICG 12625 sucrose-rich. This study also provided insights into the molecular basis of sucrose metabolism during seed development and facilitated exploring key candidate genes and molecular breeding for sucrose content in peanuts.

Sucrose-phosphate phosphatase from sugarcane reveals an ancestral tandem duplication

BackgroundSugarcane is capable to store large amounts of sucrose in the culm at maturity hence it became a major source of sucrose for the food and the renewable energy industries. Sucrose, the main disaccharide produced by photosynthesis, is mainly stored in the vacuole of the cells of non-photosynthetic tissues. Two pathways are known to release free sucrose in plant cells, one is de novo synthesis dependent on sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (S6PP) while the other is regulatory and dependent on sucrose synthase (SuSy) activity. The molecular understanding of genes that give rise to the expression of the enzyme sucrose phosphate phosphatase, responsible for the release of sucrose in the last synthetic step lag behind the regulatory SuSy gene.ResultsSugarcane genome sequencing effort disclosed the existence of a tandem duplication and the present work further support that both S6PP.1 and S6PP_2D isoforms are actively transcribed in young sugarcane plants but significantly less at maturity. Two commercial hybrids (SP80–3280 and R570) and both Saccharum spontaneum (IN84–58) and S.officinarum (BADILLA) exhibit transcriptional activity at three-month-old plants of the tandem S6PP_2D in leaves, culm, meristem and root system with a cultivar-specific distribution. Moreover, this tandem duplication is shared with other grasses and is ancestral in the group.ConclusionDetection of a new isoform of S6PP resulting from the translation of 14 exon-containing transcript (S6PP_2D) will contribute to the knowledge of sucrose metabolism in plants. In addition, expression varies along plant development and between sugarcane cultivars and parental species.

Differential expression in leaves of Saccharum genotypes contrasting in biomass production provides evidence of genes involved in carbon partitioning

BackgroundThe development of biomass crops aims to meet industrial yield demands, in order to optimize profitability and sustainability. Achieving these goals in an energy crop like sugarcane relies on breeding for sucrose accumulation, fiber content and stalk number. To expand the understanding of the biological pathways related to these traits, we evaluated gene expression of two groups of genotypes contrasting in biomass composition.ResultsFirst visible dewlap leaves were collected from 12 genotypes, six per group, to perform RNA-Seq. We found a high number of differentially expressed genes, showing how hybridization in a complex polyploid system caused extensive modifications in genome functioning. We found evidence that differences in transposition and defense related genes may arise due to the complex nature of the polyploid Saccharum genomes. Genotypes within both biomass groups showed substantial variability in genes involved in photosynthesis. However, most genes coding for photosystem components or those coding for phosphoenolpyruvate carboxylases (PEPCs) were upregulated in the high biomass group. Sucrose synthase (SuSy) coding genes were upregulated in the low biomass group, showing that this enzyme class can be involved with sucrose synthesis in leaves, similarly to sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). Genes in pathways related to biosynthesis of cell wall components and expansins coding genes showed low average expression levels and were mostly upregulated in the high biomass group.ConclusionsTogether, these results show differences in carbohydrate synthesis and carbon partitioning in the source tissue of distinct phenotypic groups. Our data from sugarcane leaves revealed how hybridization in a complex polyploid system resulted in noticeably different transcriptomic profiles between contrasting genotypes.

Regulation Network of Sucrose Metabolism in Response to Trivalent and Hexavalent Chromium in Oryza sativa.

The presence of chromium (Cr) in cultivated fields affects carbohydrate metabolism of rice (Oryza sativa L.) and weakens its productivity. Little is known about the molecular mechanism of sucrose metabolism underlying Cr stress response in rice plants. In the present study, the transcriptome map of sucrose metabolism in rice seedlings exposed to both trivalent and hexavalent chromium was investigated using Agilent 4 × 44K rice microarray analysis. Results indicated that Cr exposure (3 days) significantly (p < 0.05) improved sucrose accumulation, and altered the activities of sucrose synthetase, sucrose phosphate phosphatase, and amylosynthease in rice tissues. We identified 119 differentially regulated genes involved in 17 sucrose metabolizing enzymes and found that gene responses in roots were significantly (p < 0.05) stronger than in shoots under both Cr(III) and Cr(VI) treatment. The network maps of gene regulation responsible for sucrose metabolism in rice plants provide a theoretical basis for further cultivating Cr-resistant rice cultivars through molecular genetic improvement.

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

Characterization of the Sucrose Phosphate Phosphatase (SPP) Isoforms from Arabidopsis thaliana and Role of the S6PPc Domain in Dimerization.

Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.

Sucrose secreted by the engineered cyanobacterium and its fermentability

The unicellular cyanobacterium, Synechococcus elongatus PCC 7942 (Syn7942), synthesizes sucrose as the only compatible solute under salt stress. A series of engineered Syn7942 strains for sucrose production were constructed. The overexpression of the native sps (encoding a natively fused protein of sucrose phosphate synthase SPS and sucrose phosphate phosphatase SPP) in Syn7942 wild type caused a 93% improvement of sucrose productivity. The strain FL130 co-overexpressing sps and cscB (encoding a sucrose transporter) exhibited a 74% higher extracellular sucrose production than that overexpressing cscB only. Both results showed the significant improvement of sucrose productivity by the double functional protein SPS-SPP. Afterwards, FL130 was cultivated under a modified condition, and the cell-free culture medium containing 1.5 g L−1 sucrose was pre-treated with an acid hydrolysis technique. Cultivated with the neutralized hydrolysates as the starting media, two widely used microorganisms, Escherichia coli and Saccharomyces cerevisiae, showed a comparable growth with that in the control media supplemented with glucose. These results clearly demonstrated that the cell-free culture of sucrose-secreting cyanobacteria can be applied as starting media in microbial cultivation.

Sucrose metabolism gene families and their biological functions.

Sucrose, as the main product of photosynthesis, plays crucial roles in plant development. Although studies on general metabolism pathway were well documented, less information is available on the genome-wide identification of these genes, their expansion and evolutionary history as well as their biological functions. We focused on four sucrose metabolism related gene families including sucrose synthase, sucrose phosphate synthase, sucrose phosphate phosphatase and UDP-glucose pyrophosphorylase. These gene families exhibited different expansion and evolutionary history as their host genomes experienced differentiated rates of the whole genome duplication, tandem and segmental duplication, or mobile element mediated gene gain and loss. They were evolutionarily conserved under purifying selection among species and expression divergence played important roles for gene survival after expansion. However, we have detected recent positive selection during intra-species divergence. Overexpression of 15 sorghum genes in Arabidopsis revealed their roles in biomass accumulation, flowering time control, seed germination and response to high salinity and sugar stresses. Our studies uncovered the molecular mechanisms of gene expansion and evolution and also provided new insight into the role of positive selection in intra-species divergence. Overexpression data revealed novel biological functions of these genes in flowering time control and seed germination under normal and stress conditions.

Sucrose phosphate synthase and sucrose phosphate phosphatase interact in planta and promote plant growth and biomass accumulation.

Bioinformatic analysis indicates that sucrose phosphate synthase (SPS) contains a putative C-terminal sucrose phosphate phosphatase (SPP)-like domain that may facilitates the binding of SPP. If an SPS-SPP enzyme complex exists, it may provide sucrose biosynthesis with an additional level of regulation, forming a direct metabolic channel for sucrose-6-phosphate between these two enzymes. Herein, the formation of an enzyme complex between SPS and SPP was examined, and the results from yeast two-hybrid experiments suggest that there is indeed an association between these proteins. In addition, in planta bioluminescence resonance energy transfer (BRET) was observed in Arabidopsis seedlings, providing physical evidence for a protein interaction in live cells and in real time. Finally, bimolecular fluorescence complementation (BiFC) was employed in an attempt to detect SPS-SPP interactions visually. The findings clearly demonstrated that SPS interacts with SPP and that this interaction impacts soluble carbohydrate pools and affects carbon partitioning to starch. Moreover, a fusion construct between the two genes promotes plant growth in both transgenic Arabidopsis and hybrid poplar.

Sucrose metabolism in halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

Sucrose accumulation has been observed in some methylotrophic bacteria utilizing methane, methanol, or methylated amines as a carbon and energy source. In this work, we have investigated the biochemical pathways for sucrose metabolism in the model halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. The genes encoding sucrose-phosphate synthase (Sps), sucrose-phosphate phosphatase (Spp), fructokinase (FruK), and amylosucrase (Ams) were co-transcribed and displayed similar expression levels. Functional Spp and Ams were purified after heterologous expression in Escherichia coli. Recombinant Spp exhibited high affinity for sucrose-6-phosphate and stayed active at very high levels of sucrose (K i =1.0±0.6M). The recombinant amylosucrase obeyed the classical Michaelis-Menten kinetics in the reactions of sucrose hydrolysis and transglycosylation. As a result, the complete metabolic network for sucrose biosynthesis and re-utilization in the non-phototrophic organism was reconstructed for the first time. Comparative genomic studies revealed analogous gene clusters in various Proteobacteria, thus indicating that the ability to produce and metabolize sucrose is widespread among prokaryotes.

Proof of concept for a novel functional screening system for plant sucrose effluxers

Membrane transporters play pivotal roles in facilitating sucrose transport in plants and their activities have been shown to impact plant growth rates and crop yield. In contrast to the well-defined mechanism of sucrose influx across plasma membranes, less is known about sucrose efflux mechanisms and the membrane proteins supporting this function. A major impediment blocking progress in this key area of plant science is the absence of a functional screening system for genes encoding sucrose effluxers. Here we report a novel yeast system for screening sucrose effluxers based on sucrose release from yeast cells genetically modified to synthesize, but not to metabolize, sucrose. Inhibiting sucrose metabolism was achieved using yeast strains, SEY 6210 and YSL4-6, carrying mutations in genes encoding invertase and maltase, respectively. Genes encoding essential components of sucrose biosynthesis, sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP), were used to transform the two yeast hosts to make strains SuPy (from SEY6210) and Ysu (from YSL4-6). Cultures of SuPy15 cells were found to be capable of synthesizing sucrose when supplied with various compounds as the sole carbon source, including non-fermentable sugars and non-sugar substrates. A proof of concept of the screening system was demonstrated by transforming SuPy15 with sucrose transporter genes known to encode plasma membrane proteins that mediate sucrose efflux. The robustness of the yeast SuPy15 system as a novel platform to screen putative plant sucrose effluxers is discussed.

Structural basis of kinetic variations in Sucrose Phosphate Phosphatase (SPP) of rice and Anabaena unveiled through computational analysis

Sucrose is an important storage form of assimilated carbon in many plant species. Unlike other sucrose biosynthetic enzymes, Sucrose Phosphate Phosphatase (SPP), the terminal enzyme in sucrose biosynthetic pathway, is the least understood. SPPs from different organisms have different kinetic properties. The current study focuses on the structural differences among SPP homologues and unveils the probable structural basis of kinetic variations. We have employed computational methods of molecular modeling and structure comparisons and identified structural variations in some of the substrate binding residues, amino acid substitutions in regions that are lining the active site and minute structural differences that can enhance the nucleophilicity of a catalytic nucleophile (Asp 9 ). We report a structurally and hence functionally important amino acid substitution (Asp 159 by Alanine) in one of the rice SPP isoforms, which can result in the disruption of a H-bond that helps in binding of sucrose at the active site of the enzyme. In this paper we discuss the structural basis of enhanced catalytic efficiency of rice SPP in comparison with a cyanobacterium (Anabaena variabilis). The natural mutations identified in our analysis of the SPP catalytic domain would be useful in re-designing the enzyme for enhanced catalytic efficiency and higher sucrose production.

Evolutionary Relationship Between Available Homologous Sequences of Sucrose Phosphate Phosphatase (SPP) Enzyme

Sucrose phosphate phosphatase (SPP) is the last enzyme of sucrose biosynthetic pathway that generates sucrose in an irreversible reaction of sucrose phosphate hydrolysis. Different photosynthetic species have different cytoplasmic sugar levels that can be attributed partly to the variation in specific activity, stability, pH specificity etc. of this key sucrose biosynthesis enzyme which in turn is due to underlying sequence variations. In an attempt to identify the extent of molecular variation we analyzed the evolutionary relationship of domains and conserved segments between its homologues at the amino acid sequence level. In this analysis, we report the differential evolution of the functionally or structurally important conserved regions in SPP. Distance based and character based comparative phylogenetic studies of full length and truncated SPP amino acid sequences revealed the evolutionary relationship between the catalytic S6PP domain and a C-terminal S6PP_C domain of unknown function. The understanding on sequence evolution provides insights to the natural design as well as the modifications that occurred at different regions of SPP during evolution. The current study is the foremost systematic approach to understand the evolutionary features in SPP homologous sequences and in future this study can be extended to unveiling the basis of minute functional variations such as specific activity, stability, pH specificity etc. among the homologues.