Structure and expression analysis of the sucrose synthase gene family in apple

Sucrose synthases(SUS) are a family of enzymes that play pivotal roles in carbon partitioning, sink strength and plant development. A total of 11 SUS genes have been identified in the genome of Malus domestica(Md SUSs), and phylogenetic analysis revealed that the Md SUS genes were divided into three groups, named as SUS I, SUS II and SUS III, respectively. The SUS I and SUS III groups included four homologs each, whereas the SUS II group contained three homologs. SUS genes in the same group showed similar structural characteristics, such as exon number, size and length distribution. After assessing four different tissues, Md SUS1 s and Md SUS2.1 showed the highest expression in fruit, whereas Md SUS2.2/2.3 and Md SUS3 s exhibit the highest expression in shoot tips. Most Md SUSs showed decreased expression during fruit development, similar to SUS enzyme activity, but both Md SUS2.1 and Md SUS1.4 displayed opposite expression profiles. These results suggest that different Md SUS genes might play distinct roles in the sink-source sugar cycle and sugar utilization in apple sink tissues.

Molecular cloning,characterization and expression profile of the sucrose synthase gene family in Litchi chinensis

Sucrose synthase(SUS,EC 2.4.1.13)is widely considered as a key enzyme involved in plant sucrose metabolism,and the gene family encoding different SUS isozymes has been identified and characterized in several plant species.However,to date scant information about the SUS genes is available in Litchi chinensis Sonn.Here,we identified five SUS genes in litchi.These Lc SUSs shared high levels of similarity in both nucleotide and amino acid sequences.Their gene structure,phylogenetic relationships,and expression profiles were characterized.Gene structure analysis indicated that the Lc SUSs have similar exon-intron structures.Phylogenetic analysis revealed that the five members could be classified into three groups(LcSUS1 and LcSUS2 in SUSⅡ,LcSUS4 and LcSUS5 in SUSⅢ,and LcSUS3 in SUSⅠ),demonstrating evolutionary conservation in the SUS family across litchi and other plant species.The expression levels of Lc SUSs were investigated via real-time PCR in various tissues and different developmental stages of aril.For tissues and organs,Lc SUSs exhibited distinct but partially redundant expression profiles in litchi,being predominantly expressed in young leaves(sink).During aril development,the expression pattern of LcSUS1 was consistent with the trend of sugar accumulation,indicating it may play important roles in determination of sink strength in aril.Moreover,transcript levels of LcSUS2,LcSUS4,and LcSUS5 varied between cultivars with different hexose/sucrose ratios,which may regulate the sugar composition in aril.Our results provide insights into physiological functions of SUS genes in litchi,especially roles in regulating sugar accumulation in aril.

Regulation of sucrose synthase activity and sugar yield by nitrogen in sugar beet

The content of sugar is influenced by sucrose synthase （SS） activity in roots. The effects of nitrogen level in the aminonitrate ratio on SS activity of leaves and roots, roots yield and sugar content in sugar beet were studied in the field experiment by nutrient solution culture. The results showed that SS activity in leaves was lower than that in roots. With nitrogen level increasing, SS decomposition activity enhanced, and synthesis activity reduced. SS activity was regulated by different nitrogen forms and the ratio of NO3 and NH4^＋. SS synthesis activity was enhanced as NH4^＋ increasing when NO3 ： NH4^＋≥ 1, and it decreased as increasing NH4^＋ when NO3 ： NH4^＋≤ 1, and it was the highest when NO3 ： NH4^＋=1. SS decomposition activity was enhanced as NO3- increasing. Sucrose content in root was lowed as nitrogen level increasing, but it was enhanced as NH4^＋ increasing in the same nitrogen level. Root and sugar yield were the highest in the medium nitrogen level and NO3 ： NH4^＋=1. The result in field experiment corresponded with that in the nutrient fluid culture. It provides a basis for using reasonably nitrogen fertilizer in sugar beet production.

Genome-wide identification and function analysis of the sucrose phosphate synthase MdSPS gene family in apple

Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

Overexpression of a Potato Sucrose Synthase Gene in Cotton Accelerates Leaf Expansion, Reduces Seed Abortion, and Enhances Fiber Production

Sucrose synthase （Sus） is a key enzyme in the breakdown of sucrose and is considered a biochemical marker for sink strength, especially in crop species, based on mutational and gene suppression studies. It remains elusive, however, whether, or to what extent, increase in Sus activity may enhance sink development. We aimed to address this question by expressing a potato Sus gene in cotton where Sus expression has been previously shown to be critical for normal seed and fiber development. Segregation analyses at T1 generation followed by studies in homozygous progeny lines revealed that increased Sus activity in cotton （1） enhanced leaf expansion with the effect evident from young leaves emerging from shoot apex; （2） improved early seed development, which reduced seed abortion, hence enhanced seed set, and （3） promoted fiber elongation. In young leaves of Sus overexpressing lines, fructose concentrations were significantly increased whereas, in elongating fibers, both fructose and glucose levels were increased. Since hexoses contribute little to osmolality in leaves, in contrast to developing fibers, it is concluded that high Sus activity promotes leaf development independently of osmotic regulation, probably through sugar signaling. The analyses also showed that doubling the Sus activity in 0-d cotton seeds increased their fresh weight by about 30%. However, further increase in Sus activity did not lead to any further increase in seed weight, indicating an upper limit for the Sus overexpression effect. Finally, based on the observed additive effect on fiber yield from increased fiber length and seed number, a new strategy is proposed to increase cotton fiber yield by improving seed development as a whole, rather than solely focusing on manipulating fiber growth.

Effect of High Temperature on Sucrose Content and Sucrose Cleaving Enzyme Activity in Rice Grain During the Filling Stage

Dynamic changes of sucrose, fructose, glucose contents and differences in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase in rice grain after flowering stage were studied under natural and high temperatures by using two japonica rice varieties Koshihikari and Sasanishiki. In rice grains, the sucrose synthase activity was higher than that of invertase, which was significantly correlated with starch accumulation rate, indicating that the sucrose synthase played an important role in sucrose degradation and starch synthesis. Under high temperature, the significant increase in grain sucrose content without any increase in fructose and glucose contents, suggested that the high temperature treatment enhanced sucrose accumulation, while diminished sucrose degradation in rice grains. Compared with the control plants, the decrease in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase with high temperature treated plants indicated that the deceleration of sucrose-degradation was related to the decrease in activities of sucrose synthase and invertase.

Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains under Two Establishment Methods

Six rice varieties, PR120, PR116, Feng Ai Zan, PR115, PAU201 and Punjab Mehak 1 were raised under aerobic and transplanting conditions to assess the effects of planting conditions on sucrose metabolising enzymes in relation to the transformation of free sugars to starch and protein in flag leaves and grains. Activities of sucrose synthase, sucrose phosphate synthase and acid invertase increased till flowering stage in leaves and mid-milky stage（14 d after flowering） in grains and thereafter declined in concomitant with the contents of reducing sugar. Under aerobic conditions, the activities of acid invertase and sucrose synthase（cleavage） significantly decreased in conjunction with the decrease in non-reducing sugars and starch content in all the varieties. Disruption of starch biosynthesis under the influence of aerobic conditions in both leaves and grains and the higher build up of sugars possibly resulted in their favoured utilization in nitrogen metabolism. Feng Ai Zan, PR115 and PR120 maintained higher levels of sucrose synthase enzymes in grains and leaves and contents of metabolites（amino acid, protein and non-reducing sugar） under aerobic conditions, while PR116, Punjab Mehak 1 and PAU201 performed better under transplanting conditions, thus showing their adaptation to environmental stress. Yield gap between aerobic and transplanting rice is attributed primarily to the difference in sink activity and strength. Overall, it appear that up-regulation of sucrose synthase（synthesis） and sucrose phosphate synthase under aerobic conditions might be responsible in enhancing growth and productivity of rice varieties.

Polyphosphate Accelerates Transformation of Nonstructural Carbohydrates to Improve Growth of ppk-Expressing Transgenic Rice in Phosphorus Deficiency Culture

Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils,a key limiting factor for sustainable development in modern agriculture.The polyphosphate kinase(ppk)gene-expressing transgenic rice with a single-copy line(ETRS)is constructed to improve phosphate fertilizer utilization efficiency for phosphorus resource conservation.To investigate the potential mechanisms of the increased biomass in ETRS in low phosphate culture,ETRS was cultivated in a low inorganic phosphate(Pi)culture medium(15μmol/L Pi,LP)and a normal Pi culture medium(300μmol/L Pi,CP),respectively.After 89 d of cultivation in different concentrations of phosphate culture media,the total phosphorus,polyphosphate(polyP),biomass,photosynthetic rate,nonstructural carbohydrate(NSC)contents,related enzyme activities,and related gene expression levels were analyzed.The results showed that ETRS had a high polyP amount to promote the photosynthetic rate in LP,and its biomass was almost the same as the wild type(WT)in CP.The NSC content of ETRS in LP was higher than that of WT in LP,but slightly lower than that of WT in CP.PolyP notably promoted the sucrose phosphate synthase activities of ETRS and significantly down-regulated the expression levels of sucrose transporter genes(OsSUT3 and OsSUT4),resulting in inhibiting the transport of sucrose from shoot to root in ETRS.It was concluded that polyP can stimulate the synthesis of NSCs in LP,which improved the growth of ETRS and triggered the biological activities of ETRS to save phosphate fertilizer.Our study provides a new way to improve the utilization rate of phosphate fertilizer in rice production.

Effects of Panicle Nitrogen Fertilization on Non-Structural Carbohydrate and Grain Filling in Indica Rice

Grain filling, a crucial stage of grain yield formation in rice, is usually affected by the panicle nitrogen （N） fertilization. Field and pot culture experiments were conducted to explore the underlying mechanisms of N effect. Two rice cultivars with high lodging resistance were grown in the field and pot. Four panicle N fertilization treatments were conducted in 2006 and repeated in 2007. The result showed that medium level of panicle N fertilization treatment （NM） enhanced the accumulation and translocation of non-structural carbohydrate （NSC） in the stem and sheath. Compared with non-nitrogen treatment （NO）, NM promoted the translocation of labeled ^13C from stem and sheath to grain. But, low level of panicle N fertilization treatment （NL） and high level of panicle N fertilization treatment （NH） showed the negative effect. The endosperm cell, grain length, and grain width of NM increased more quickly than that of NO from 4 to 10 d after anthesis. During the early period of grain filling, sucrose-phosphate synthase （EC 2.4.1.14, SPS） activity were significantly higher for the NM treatment than those of the NL and NH treatments. Sucrose synthase （EC 2.4.1.13, SuSase） activity in the grains was substantially enhanced by NM, with the duration of higher activity being longer than those of the other treatments. At maturing stage, NM significantly increased the filled grain number, the seed-setting rate, and the grain weight compared with NL and NH. The results suggest that NM have a positive effect on the activities of enzymes of physiological importance, thereby increasing the grain size and promoting grain filling.

Effect of Nitrogen on Apple Fruit Development in Different Loads

The experiments including soil application N, thinning and priming leaf treatment, using eight -year-old apple trees (M. domestica Borkh.cv.Red Fuji/ M. hupenensis Rhed) as materials were carried out to study the effect of N on fruit development. The main results were as follows: on heavily thinned trees, SS activity was independent of N and priming leaves treatments. The results show that the carbohydrate restriction of source to sink activity did not exist, and N application did not stimulate fruit growth rates relative to those on nonfertilized trees; however, N fertilization resulted in a longer fruit development period and increased the growth potential of individual fruit by 20.8% (fresh weight) and 14.1% (dry weight) vs. controls ; in unthinned trees, SS activity was increased by N fertilization but decreased by priming leaves treatment , so the carbohydrate restriction of source to sink activity existed, N fertilization increased the average single fruit weight both by extending the fruit development period and by increasing fruit growth rate, and the increasing rates were 28.2% (fresh weight) and 19.4 % (dry weight) compared to the unthinned nonfertilized controls. Fruit soluble sugar and pericarp anthocyanin concentration was decreased by N fertilization.

Environmentally Induced Under-Development of Seeds in Garden Pea and Its Underlying Factors

Under-development of pea (Pisum sativum L.) seeds in normally developed pods, resulting in “unfilled pods”, has been a serious problem in the greenhouse cultivation in Wakayama Prefecture, one of the major regions of pea cultivation in Japan. This phenomenon appears to be induced by low temperature and low solar irradiation during pea growing season. This study examined the relationship between this seed under-development and sucrose-to-starch metabolism in developing seeds and pods after flowering. The starch content, ADP-glucose pyrophosphorylase (AGPase) and sucrose synthase activities of a pea cultivar, Kishuusui, were monitored through seed development in shading-treatment plot and in control plot. Results showed that the present treatment induced the depression of starch accumulation and AGPase activity in developing seeds compared with control, which might cause the occurrence of under-development of seeds and unfilled pods. Surplus carbohydrates from source organs might be stored in pod walls, as expected from higher starch level and AGPase activity in treated pod walls. The necessity of thermostable AGPase variants in pea to prevent the unfilled-pod problem was discussed.

Recovery of photosynthesis,sucrose metabolism,and proteolytic enzymes in Kandelia obovata from rare cold events in the northernmost mangrove,China

Introduction:Understanding how mangroves respond to rare cold events has implications for both restoration and conservation under climate change scenarios.This study investigated the responses of photosynthesis and activities of key enzymes involving carbon and nitrogen metabolism at different ages of Kandelia obovata to a rare cold event in the winter of 2010.Methods:This study took place on Ximen Island,Zhejiang Province,China.We measured the physiological recovery of 2-3-,5-6-,9-10-and 54-55-year-old K.obovata trees after freezing injury in February and March in 2011 and 2012,respectively.Results:Chilling injury index and electrolyte leakage of K.obovata increased with increasing tree age in the winter of 2010,and electrolyte leakage in K.obovata at different ages in the winter of 2010 was far higher than that in the winter of 2011.The rare cold events significantly changed the recoveries of the leaf net photosynthetic rate(Pn)and stomatal conductance(Gs);ratios of chlorophyll a/chlorophyll b(Chl a/Chl b);contents of total soluble sugar(TSS),sucrose,free amino acid(FAA),and soluble protein;and activities of sucrose phosphate synthase(SPS),endopeptidase,and carboxypeptidase in K.obovata at different ages.These effects were mainly due to changes in the physiological mechanism in the 2-year-old trees.A clear decrease in Pn of the 2-year-old trees was observed in February 2011,as exemplified by reductions in ratios of Chl a/Chl b and chlorophyll/carotenoid(Chl/Car),as well as inhibition of the levels of TSS and FAA(osmotic substances).During recovery in 2011 and 2012,the activities of SPS and sucrose synthase(SS)were responsible for sucrose synthesis after the rare cold events in 2011,but only SPS activity was one of the main factors contributing to the metabolism of stachyose to sucrose without cold damage in 2012.Carboxypeptidase played a more important role than endopeptidase during protein hydrolysis after the rare cold events.Conclusions:The results suggest that the recovery of photosynthetic capacity in K.obovata was changed after a rare cold event,which is associated with pigment components and activities of SS,SPS,and carboxypeptidase,especially the seedlings.

Alterations in sucrose metabolizing enzyme activities and total phenol content of Curcuma longa L. as affected by different triazole compounds

Changes in the sucrose metabolism of Cur-cuma longa L.plants were studied under treatment with different triazole compounds viz.,triadimefon(TDM)and propiconazole(PCZ).Plants were treated with TDM at 15mg/L and PCZ at 10mg/L separately by soil drenching on 80,110,and 140 days after planting(DAP).The plants were harvested randomly on 90,120,and 150DAP to determine the effect of both the triazoles on sucrose metabolizing enzymes and phenol content.The sucrose metabolism was studied by analyzing sucrose metaboliz-ing enzymes like sucrose synthase and sucrose phosphate synthase.All the analyses were assayed in leaves and tubers of both control and treated plants.It was found that both of the triazole compounds had profound effects on these parameters.

Genome-Wide Analysis of the Sus Gene Family in Cotton

Sucrose synthase （Sus） is a key enzyme in plant sucrose metabolism. In cotton, Sus （EC 2.4.1.13） is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group （Sus I） underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members＇ physiological functions during fiber growth and development.

Carbon Supply and the Regulation of Cell Wa Synthesis

All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent the largest sink for photosynthetically fixed carbon, both for individual plants and in the terrestrial biosphere as a whole. Cell wall synthesis is a highly sophisticated process, involving multiple enzymes and metabolic intermediates, intracellular trafficking of proteins and cell wall precursors, assembly of cell wall polymers into the extracellular matrix, remodeling of polymers and their interactions, and recycling of cell wall sugars. In this review we discuss how newly fixed carbon, in the form of UDP-glucose and other nucleotide sugars, contributes to the synthesis of cell wall polysaccharides, and how cell wall synthesis is influenced by the carbon status of the plant, with a focus on the model species Arabidopsis （Arabidopsis thaliana）.

Evidence for the Role Evolutionary Increase Yield in Cotton of Transfer Cells in the in Seed and Fiber Biomass

Transfer cells （TCs） are specialized cells exhibiting invaginated wall ingrowths （Wls）, thereby amplifying their plasma membrane surface area （PMSA） and hence the capacity to transport nutrients. However, it remains unknown as to whether TCs play a role in biomass yield increase during evolution or domestication. Here, we examine this issue from a comparative evolutionary perspective. The cultivated tetraploid AD genome species of cotton and its A and D genome diploid progenitors displayed high, medium, and low seed and fiber biomass yield, respectively. In all three species, cells of the innermost layer of the seed coat juxtaposed to the filial tissues trans-differentiated to a TC morphology. Electron microscopic analyses revealed that these TCs are characterized by sequential formation of flange and reticulate Wls during the phase of rapid increase in seed biomass. Significantly, TCs from the tetraploid species developed substantially more flange and reticulate Wls and exhibited a higher degree of reticulate WI formation than their progenitors. Consequently, the estimated PMSA of TCs of the tetraploid species was about 4 and 70 times higher than that of TCs of the A and D genome progenitors, respectively, which correlates positively with seed and fiber biomass yield. Further, TCs with extensive Wls in the tetraploid species had much stronger expression of sucrose synthase, a key enzyme involved in TC Wl formation and function, than those from the A and D progenitors. The analyses provide a set of novel evidence that the development of TC Wls may play an important role in the increase of seed and fiber biomass yield through polyploidization during evolution.

G-Quadruplex(G4) Motifs in the Maize(Zea mays L.) Genome Are Enriched at Specific Locations in Thousands of Genes Coupled to Energy Status,Hypoxia,Low Sugar,and Nutrient Deprivation

The G-quadruplex （G4） elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This （34 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationaily identified 149,988 non-telomeric G4 motifs in maize （Zea mays L., B73 AGPv2）, 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5~ UTR and the other at the 5~ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia （group VII ERFs）, oxidative stress （D J-1/GATasel）, and energy status （AMPK/ SnRK） pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-l-related protein kinase 1 （SnRK1） has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKIN/β1 expression was reduced upon sugar treatment （including mannitol） and did not affect the activity of sucrose phosphate synthase. The results indicate that AKIN/β1 is involved in the regulation of nitrogen metabolism and sugar signaling.