Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Cloning and Expression of the Promoter of Maize Starch-Branching Enzyme sbeⅡb Gene

[Objective] The aim was to construct promoter of maize starch-branching enzyme sbe Ⅱb gene specifically expressed in seed.[Method] The promoter sequence of maize starch-branching enzyme sbe Ⅱb gene was amplified by LA-PCR and then cloned into pMD18-T vector.Subsequently,the promoter was cloned into pBI121 vector to construct plant expression vector pBI121-sbe Ⅱb.Recombinant plasmid pSBE-GUS was constructed by connecting GUS gene into pBI121-sbe Ⅱb and transformed into tobacco mediated by Agrobacterium tumefaciens,before detection by PCR and Southern blot.The biochemical analysis and fluorescence detection of GUS activity were performed in different parts of the tobacco by Gene Gun Method and Agrobacterium tumefaciens transformation respectively.[Result] The homology between promoter sequence cloned in this experiment and the corresponding sequence announced in the GenBank reached 98.52%.Four transformed tobaccos were obtained by PCR and Southern blot after co-culture and selective culture.A small number of blue spots appeared in leaves,and the spots could barely been seen in the stems and roots,while a large number of blue spots were found in seeds.So,it could be concluded that the promoter of sbeⅡb gene was specifically expressed in seeds.[Conclusion] Promoter of maize starch branching enzyme sbe Ⅱb specifically expressed in seeds was successfully cloned.

Effects of Weak Light on Starch Accumulation and Starch Synthesis Enzyme Activities in Rice at the Grain Filling Stage

Dynamic changes of starch, amylose, sucrose contents and the activities of starch synthesis enzymes under shading treatments alter flowering were studied using two rice varieties IR72 （indica） and Nipponbare （japonica） as materials. Under shading treatments, the starch, amylose and sucrose contents decreased, while ADP-glucose pyrophosphorylase （ADPGPPase） activity only changed a little, soluble starch synthase activity and granule bound starch synthase activity decreased, soluble starch branching enzyme （SSBE, Q-enzyme） activity and granule bound starch branching enzyme （GBSBE, Q-enzyme） activity increased, and starch debranching enzyme （DBE, R-enzyme） activity varied with varieties. Correlation analyses showed that the changes of starch content were positively and significantly correlated with the changes of sucrose content in the weak light. Both ADPGPPase activity and SSBE activity were positively and significantly correlated with starch accumulation rate. It was implied that the decline of starch synthase activities was related to the decrease of starch content and the increase of the activity of starch branching enzyme played an important role in the decrease of the ratio of amylose to the total starch under the weak light.

Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme （SBE） Ⅰ and Ⅱ, respectively, in sweet potato （Ipomoea batatas L.） were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid （ABA） was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

Changes in Starch Accumulation and Activity of Enzymes Associated with Starch Synthesis of Rice at Different N Supplying Dates

The changes in grain-filling, starch accumulation and activity of enzymes associated with starch synthesis in two different hybrid rice varieties were analyzed at different N supplying dates (earlier-date-emphasized, mean-date-emphasized and later-date- emphasized). The results showed that the N application of later-date-emphasized could promote grain-filling rate, increase grain weight and amylopectin content. The peak of activity in three enzymes of ADPglusoce pyriphosphorylase (ADPG), starch synthesis enzyme (SSS) and starch branching enzyme (SBE) in grains of two different rice varieties was not changed obviously, but the mean and maximum activity of these three enzymes changed, and the changes of SSS and ADPG were bigger than that of SBE as N supplying date changed. The N application of earlier-date-emphasized increased SSS activity and the N application of later-date-emphasized increased ADPG and SBE activities. The mean SSS activity during whole grain-filling period, and ADPG and SBE activities at middle and late period of grain-filling were significantly or very significantly correlated with grain-filling rate and accumulating rate of amylose and amylopectin. Both of ADPG and SBE played an equal important role in the changes of amylose and amylopectin content. The N application of later-date-emphasized increased amylose and amylopectin accumulating rate.

Effect of Temperature at Grain Filling Stage on Activities of Key Enzymes Related to Starch Synthesis and Grain Quality of Rice

Three japonica rice varieties with different cooking and eating quality were grown at high temperature in the greenhouse and natural field. Effects of temperature at the grain filling stage on these varieties were investigated in terms of the activities of key enzymes related to starch synthesis and cooking and eating quality of rice grain. The high temperature at the grain filling stage increased protein content, and decreased amylose content and taste meter value of rice; inferior grain quality varieties showed a greater magnitude of the increase or decrease than the superior ones. Reaction of rapid visco analyser profiles to the temperature varied with rice varieties. The activities of adenosine diphosphoglucose pyrophosphoryiase （AGPP）, soluble starch synthase （SSS） and starch branching enzyme （SBE） gradually increased to a peak value, and thereafter declined as grain filling progressed. Enzyme activities in different varieties differed in a same filling stage, and also in the time when the enzyme activity reached a maximum. AGPP and SSS were insensitive to the environmental temperature, but SBE was comparatively sensitive to the temperature, and its activity declined when temperature was too high or too low.

Effects of Early- and Late-Sowing on Starch Accumulation and Associated Enzyme Activities During Grain Filling Stage in Rice

The environmental temperature occurring during the grain filling stage is an important factoraffecting starch synthesis and accumulation in rice. We investigated starch accumulation, amylaseactivity and starch granule size distribution in two low-amylose japonica rice varieties, Nanjing 9108 andFujing 1606, grown in the field at different filling temperatures by manipulating sowing date. The two ricevarieties exhibited similar performances between two sowing dates. Total starch, amylose andamylopectin contents were lower at the early-filling stage of T1 treatment (Early-sowing) compared withthose at the same stage in T2 treatment (Late-sowing). In contrast, at the late-filling stage, when fieldtemperatures were generally decreasing, total starch and amylopectin contents in T1 were highercompared to those in T2. The ideal temperature for strong activity of ADP-glucose pyrophosphorylaseand soluble starch synthase was about 22℃. A higher temperature from the heading to maturity stagesin T1 increased the activities of starch branching enzyme and suppressed the activities of granule boundstarch synthetase and starch debranching enzyme. We found that rice produced larger-sized starchgranules under the T1 treatment. These results suggested that due to the early-sowing date, the hightemperature (30℃) occurring at the early-filling stage hindered starch synthesis and accumulation,however, the lower temperatures (22 ℃) at the late-filling stage allowed starch synthesis and accumulationto return to normal levels.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Relationship Between Variation in Activities of Key Enzymes Related to Starch Synthesis During Grain Filling Period and Quality of Eating and Cooking in Rice

Four japonica rice varieties with significant differences in quality of eating and cooking were used in the experiment. The varieties showed differences in amylose and amylopectin contents at different grain filling stages, which were attributed to the accumulative speed of starch at different grain filling stages. During grain filling period, the varieties had no difference in the time when the activities of ADPglocose pyrophosphorylase （AGPP） and soluble starch synthesis （SSS） reached a maximum, but had difference in the time when the activity of starch branching enzyme （SBE） reached a maximum, in which the inferior quality varieties were earlier than the high quality ones, and high quality varieties still kept high enzyme activities at the late stage of grain filling. The correlation and correlative degree between AGPP, SSS, SBE and amylose content, amylopectin content, taste meter value, and RVA properties varied with the different stages of grain filling. The correlation between SSS activity and taste meter value was not significant during the whole period of grain filling, but the activities of AGPP and SBE had significant or highly significant correlation with taste meter value. It was helpful for improving quality of eating and cooking of japonica rice to use the materials with low enzyme activity at the early stage of grain filling or high enzyme activity at the late stage as parents.

The difference of soluble sugar contents and starch synthetic key enzyme activities between high and low starch cultivar of cassava

The difference of soluble sugar contents and starch synthetic key enzyme activities between high starch cultivar and low starch cultivar were studied in this experiment, of which FuXuan01, GR891, SC124, and SC201 were used as materials. The results showed that the contents of reducing sugar in root tube of low starch cultivars were higher than those of high starch cultivars in all growth periods, the contents of sucrose in root tube of high starch cultivar were higher than those of low starch cultivars in the early growth period, but, lower in the late growth period, and there were almost no difference in the contents of soluble sugar in root tube between high starch cultivar and low starch cultivar in the early growth period, but notable difference in the late growth period and the soluble sugar contents of low starch cultivars were higher than those of high starch cultivars. It also-showed that the activities of ADPGPpase, SSS, and SBE （starch branching enzyme） of high starch cultivars were higher than those of low starch cultivars. It was evident that there were close correlations between the content of sucrose, reducing sugar and soluble sugar in root tube, and the activities of ADPGPpase, SSS, SBE, and the starch accumulation in the root tubers of cassava. It was quite evident in this experiment that the soluble sugar and starch synthetic key enzyme were main factors controlling starch accumulation in root tubers. These results provided important indication for physiological controlling in high starch cultivation and gene engineering breeding of high starch cultivar of cassava.

Origin and evolution of the main starch biosynthetic enzymes

Starch,a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications.Despite the starch biosynthetic pathway’s main enzymes have been characterized,their origin and evolution remained a subject of debate.In this study,we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes:starch synthase(SS),starch branching enzyme(SBE)and isoamylase-type debranching enzyme(ISA)from 51,151 annotated genomes.Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway.Initially,the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor(LECA)via horizontal gene transfer(HGT).This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen.Furthermore,during the emergence of Archaeplastida,one clade of SS was transferred from Deltaproteobacteria by HGT,while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer(EGT).Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway.Subsequently,after the divergence of Viridiplantae from Rhodophyta,all three enzymes underwent multiple duplications and N-terminus extension domain modifications,resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway.By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway,this study provides important insights into the evolutionary events of plants.

Effects of laponite and silica nanoparticles on the cleaning performance of amylase towards starch soils

This work aims to understand the effect of nanoparticle-enzyme interactions and how such interactions affect starch based soil removal. Silica and laponite are used as the model nanoparticles, and s-amylase is employed as the model enzyme. The results show that, if the nanoparticles and enzyme are added simultaneously, laponite enhances the enzyme performance toward starch soil removal, whereas silica imposes a small effect on the enzymatic activity towards the same soil substrates. However, when nanoparticles are added first, the enzyme activity is not affected much by laponite but is hindered significantly by silica nanoparticles. Furthermore, sequential addition of the enzyme followed by silica nanoparticles improves soil removal. Electron microscopic analyses, measurements of the enzyme activity in suspen- sions of nanoparticles, and particle size characterisation suggest that dense coverage of soil surface by the silica nanoparticles be likely a mechanism for the experimentally observed hindrance of soil removal when silica nanoparticles are added before enzyme.