GBSS Activity Research Articles

Relationships between the appearance quality and starch structure of soft rice under different nitrogen levels

AbstractThis study aims to explore the formation mechanism of starch structure and the relationships between the appearance quality and starch structure of soft rice under different nitrogen levels. We comprehensively investigated the physiological aspects, starch structure variations, and appearance quality of soft rice in response to different nitrogen applications. The results showed that under the moderate nitrogen application (270 N), the soft rice exhibited the highest AGPase activity, the highest large‐starch granule content, and the lowest chalkiness. Under the highest nitrogen application (360 N), the soft rice exhibited the highest GBSS and DBE activity and the lowest SBE activity, the highest content of long‐branched amylopectin, the lowest relative crystallinity, the fewest ordered structures, the most amorphous structures, the largest semi‐crystalline lamellar thicknesses, and the highest transparency of chalk‐free rice. In conclusion, moderate nitrogen fertilization (270 N) improved the AGPase activity, which leaded to fuller starch granules and more compact endosperm in soft rice. Thus, the grain chalkiness of soft rice decreased. Continuous nitrogen application (0‐360 N) constantly increased the GBSS and DBE activity and reduced the SBE activity in soft rice, leading a lower content of short‐branched amylopectin and a higher content of long‐branched amylopectin in soft rice. Thus, the relative crystallinity and ordered structures of soft rice were reduced. These structures improved the transparency phenotype of soft rice.

Metabolomic study on the quality differences and physiological characteristics between rice cultivated in drought and flood conditions

The differences between dry- and flood-cultivated rice and the reason behind low-quality dry-cultivated rice were clarified. The physiological traits, starch synthase activity, and grain metabolomics of ‘Longdao 18′ were measured and analyzed at four growth stages. The brown, milled, and whole-milled rice rates and AGPase, SSS, and SBE activity were lower after drought treatment than during flood cultivation, while the chalkiness, chalky grain rate, amylose (16.57–20.999%), protein (7.99–12.09%), and GBSS activity were higher. Related enzymatic gene expression showed significant differences. Metabolic results showed pyruvate, glycine, and methionine upregulation at 8DAF and higher citric, pyruvic, and α-ketoglutaric acid content at 15DAF. Therefore, 8DAF-15DAF represented the crucial quality formation period for dry-cultivated rice. At 8DAF, the respiratory pathways used amino acids as signaling molecules and alternative substrates to adapt to energy shortages, arid environments and rapid protein accumulation and synthesis. Excessive amylose synthesis at 15DAF accelerated reproductive growth, promoting rapid premature aging.

Response of Rice with Overlapping Growth Stages to Water Stress by Assimilates Accumulation and Transport and Starch Synthesis of Superior and Inferior Grains.

Drought stress at jointing–booting directly affects plant growth and productivity in rice. Limited by natural factors, the jointing and booting stages of short-growth-period rice varieties are highly overlapped in high-latitude areas, which are more sensitive to water deficit. However, little is known about the dry matter translocation in rice and the strategies of starch synthesis and filling of superior and inferior grains under different drought stress was unclear. In this study, the rice plants were subjected to three degrees of drought stress (−10 kPa, −25 kPa, −40 kPa) for 15 days during the jointing–booting stage; we investigated dry matter accumulation and translocation, grain filling and enzyme activities to starch synthesis of superior and inferior grains in rice with overlapping growth stages from 2016 to 2017. The results showed that drought stress significantly reduced dry matter accumulation in the stems and leaves. Mild and moderate drought increased dry matter translocation efficiency. However, severe drought stress largely limited the dry matter accumulation and translocation. A large amount of dry matter remains in vegetative organs under severe drought stress. The high content in NSC in stem and sheath plays a key role in resisting drought stress. The drought stress at jointing–booting directly caused a change in the grain filling strategy. Under moderate and severe drought, the grain-filling active period of the superior grains was shortened to complete the necessary reproductive growth. The grain-filling active period of the inferior grains was significantly prolonged to avoid a decrease in grain yield. The significant decrease in the grain-filling rate of the superior and inferior grains caused a reduction in the thousand-grain weight. In particular, the influence of the grain-filling rate of inferior grains on the thousand-grain weight was more significant. Drought stress changed the starch synthesis strategies of the superior and inferior grains. Soluble starch synthase and starch branching enzyme activities of inferior grains increased significantly under drought stress. GBSS activity was not sensitive to drought stress. Therefore, amylose content was decreased and amylopectin synthesis was enhanced under drought stress, especially in inferior grains.

Dynamics of starch synthesis enzymes and their relationship with chalkiness of early indica rice under different postanthesis temperature regimes

An experiment on an early indica rice cv. ‘Shenyou9576’ was conducted in the Key Laboratory of Phytohormones and Growth Development of Hunan Agricultural University, Changsha, Hunan, PR China in 2014 to investigate the influence of varying post-anthesis temperatures on chalkiness rate, head rice rate, and on major 6 starch synthesis enzymes i.e., SuSy (EC 1.9.3.1), ADPG-Ppas (EC 2.7.7.27), SSS (EC 2.4.1.21) and GBSS, (EC 2.4.1.21), SBE (EC 2.4.1.18) and SDBE (EC 3.2.1.70). The treatments comprised of three temperature regimes which are designated as the high (35/28oC- day/night), low (25/20oC- day/night) and natural condition (35/25oC-day/night) as the control. Under high temperature maximum chalkiness rate was 61.11% and minimum was 22.59% under low temperature treatment. The lowest head rice rate was 42.76% under high temperature treatment followed by 49.91% in the control, while the highest rate was 62.33% under low temperature treatment. Maximum grain filling rate (Gmax) was found highest (1.69 mg/day) in the high temperature and average grain filling rate (Gavg) was found highest (1.36 mg/day) under the control. The activity of SuSy, ADPG-Ppase, SSS and GBSS were decreased gradually from 14 to 35 days after flowering (DAF). Irrespective of the treatments, an increasing trend of ADPG-Ppase activity was observed from 7 to 14 DAF and then declined. Correlation between the chalkiness and the enzymes activity of SuSy, ADPG-Ppase and SSS were significantly negative at 21, 28 and 35 DAFs, i.e., higher activity of SuSy, ADPG-Ppase and SSS at the mid-late to the late caryopsis development stage mediated by low temperature treatment played an important role for the reduction of chalkiness. The correlation between GBSS activity and chalkiness was significantly negative and stronger at 14, 21 and 28 DAF indicating that GBSS played a cardinal role to reduce chalkiness in the mid to mid-late stage of rice grain development. Significantly negative correlation was found between starch branching enzyme (SBE) and chalkiness at 21, 28 and 35 DAF, i.e., the higher SBE activity under low temperature treatment at the later grain filling stage also had a positive role in reduction of chalkiness.
 Bangladesh J. Agril. Res. 44(2): 223-238, June 2019

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice.

SummaryAmylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT‐HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the approach of sequencing bulks was able to identify genes on different chromosomes associated with this complex trait.

Physicochemical properties of starches and expression and activity of starch biosynthesis-related genes in sweet potatoes

The functional properties of starches from six sweet potato varieties containing various starch components and structures were studied in an attempt to identify starch sources for industrial uses. Tainan 18 (TNN18) with high-amylose (AM) starch exhibited high setback and breakdown viscosities, high water solubility at 85°C but low swelling volume at 65°C, and high hardness and adhesiveness; in contrast, the low-AM starch of Tainung 31 (TNG31) had opposite characteristics. Seven genes related to starch biosynthesis were tested, and GBSS, SS, SBEII, ISA, and AGPase were highly expressed in TNN18 and TNG31; however, transcript levels in DBE and SBE were extremely low. GBSS and SS activity reflected the abundance of GBSS and SS mRNA in TNG31 and TNN18, and expression of AGPase, GBSS, SS, and SBE in TNN18 substantially increased content of AM. The expression and activity of DBE had a significant effect on TNG31 with increased AP content.

Effect of Temperature on Starch Accumulation and Activities of Starch Synthetic Key Enzymes during Grain-Filling Stage of Aromatic Rice

Changes of the content of starch, amylose and the activities of 3 key enzymes involved in starch synthetic metabolism in developing rice grains under four different temperature in phytotrons during grain filling stage of two aromatic rice varieties were studied. The results showed that compared with that under the lower temperature (21°C and 23°C), the content of starch and amylose and the activities of SBE and GBSS under higher temperatures (26°Cand 30°C) were generally higher at the beginning of the grain filling stage and obviously lower during the middle and late grain filling stage, with the exception of the activity SSS which were always higher under lower temperature. The relationship between the changes of activities of SSS, SBE and the variation of starch accumulating rate was very close and so did the relationship between GBSS activity and the amylose content. The lower activities of SSS, SBE and GBSS during the middle and late grain filling stage may be the key physiological factors which lead to the poorer progress quality and appearance quality of aromatic rice under high temperature.

Analysis of Amylose Accumulation During Seed Development in Maize

Starch, which includes amylose and amylopectin, is the most important component in maize ( Zea mays L.) seeds. The accumulation of amylose in maize seeds was examined in this study. The percentage of amylose content gradually increased in seeds from day 10 to day 25 after pollination, which is consistent with the changes of GBSS activity. The transcripts of GBSSI were detected in both the endosperm and embryo of wild-type maize. However, its transcripts, GBSS activity, and amylose were not detected in either the endosperm or embryo of waxy maize. These results indicate that the accumulation of amylose is controlled by GBSSI expression in the seeds of maize.

Granule-bound starch synthase cDNA in Chlorella kessleri 11 h: cloning and regulation of expression by CO2 concentration

The cDNA for the granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) of Chlorella kessleri 11 h was isolated and characterized. CkGBSS encodes a 609-amino acid polypeptide (65,627 Da) that includes an N-terminal hydrophobic signal peptide of 55 amino acids. The deduced amino acid sequence of the mature CkGBSS polypeptide shares a greater identity (65%) to that of the GBSS protein of Chlamydomonas reinhardtii, than to those of vascular plant species, but does not have the extra-long C-terminal sequence found in C. reinhardtii. When CO(2 )concentration was decreased from 3 to 0.04% (air level) in light, the levels of CkGBSS mRNA, CkGBSS protein, and GBSS activity increased. Under this condition, pyrenoid and pyrenoid starch developed, and the relative amount of amylose in starch increased. These observations suggest that low CO(2) level up-regulates GBSS biosynthesis at the transcriptional level.

The isolation and characterization of a waxy mutant of diploid wheat ( Triticum monococcum L.)

A waxy mutant of diploid wheat ( Triticum monococcum L.) was isolated by screening M 3 seeds derived from 1% ethyl methanesulfonate (EMS) mutagenized materials with KI-I 2 staining of endosperm starch. This mutant was controlled by a single waxy gene that completely lacked amylose, the 59-kDa waxy protein and the granule-bound starch synthase I (GBSS I) activity in the endosperm. Moreover, the T. monococcum exhibited the gene dosage effects in terms of amylose content, the amount of waxy protein and the GBSS activity. The chain length distribution of amylopectin of the waxy mutant endosperm starch slightly differed from that of the wild type; the long chains of DP≧19 were more enriched in the waxy amylopectin than those in the wild type. The mutant can be an important material for studies on starch metabolism and breeding in Triticum species.

Expression of a cassava granule‐bound starch synthase gene in the amylose‐free potato only partially restores amylose content

ABSTRACTGranule‐bound starch synthase I (GBSS I) is responsible for the synthesis of amylose in starch granules. A heterologous cassava GBSS I gene was tested for its ability to restore amylose synthesis in amylose‐free (amf) potato mutants. For this purpose, the cassava GBSS I was equipped with different transit peptides. In addition, a hybrid containing the potato transit peptide, the N‐terminal 89 amino acids of the mature potato GBSS I, and the C‐terminal part of cassava GBSS I was prepared. The transgenic starches were first analysed by iodine staining. Only with the hybrid could full phenotypic complementation of the amf mutation be achieved in 13% of the plants. Most transformants showed partial complementation, but interestingly the size of the blue core was similar in all granules derived from one tuber of a given plant. The amylose content was only partially restored, up to 60% of wild‐type values or potato GBSS I‐complemented plants; however, the GBSS activity in these granules was similar to that found in wild‐type ones. From this, and the observation that the hybrid protein (a partial potato GBSS I look‐alike) performs best, it was concluded that potato and cassava GBSS I have different intrinsic properties and that the cassava enzyme is not fully adapted to the potato situation.

A 56-kDa protein is a novel granule-bound starch synthase existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat (Triticum monococcum L.).

A novel 56-kDa granule-bound starch synthase (GBSS; NDPglucose-starch glucosyltransferase, EC 2.4.1.21) responsible for amylose synthesis was found in the pericarps, aleurone layers and embryos of immature diploid wheat (Triticum monococcum L.). The GBSS and other proteins bound to starch granules of various tissues of immature normal and waxy diploid wheat seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their activities were examined. In the waxy mutant, the waxy protein (59.5 kDa, GBSSI) was absent, but amylose and GBSS activity were evident in all tissues except the endosperm. Of the proteins bound to starch granules, only the 56-kDa protein was associated with the presence of amylose and GBSS activities in the pericarps, aleurone layers and embryos. Mutations at the waxy locus did not affect the 56-kDa protein in these tissues. Changes in the amount of 56-kDa protein during the course of seed development, and the distribution of the 56-kDa protein in each tissue of immature seeds were quite different from those of the waxy protein. On the other hand, the N-terminal amino acid sequence of the 56-kDa protein had a 40-50% similarity to GBSSI of some other plant species and was antigenically related to the waxy protein. These results strongly suggest that the 56-kDa protein in diploid wheat is a GBSSI class enzyme and, hence, an isoform of the waxy protein. The waxy protein and 56-kDa protein, however, are expressed in different seed tissues and at different stages of seed development.

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

A gene-dosage population was obtained by crossing two genotypes that were duplex for the GBSS allele. Nulliplex, simplex, duplex or triplex/quadruplex plants could be identified by monitoring the segregation of red and blue microspores after staining with iodine. GBSS activity was significantly different for all groups and showed an almost linear dosage effect for the wildtype GBSS gene. A dosage effect was found for amylose content that was not linear. The amylose content was similar for both the duplex and triplex/quadruplex group. Within the simplex group, differences in amylose content were found, which might be due to a different genetic background. There was no linear correlation between GBSS activity and amylose content. A certain level of GBSS activity led to a maximum amount of amylose, and further increase in GBSS activity did not result in a further increase in amylose content. The presence of one or more wildtype GBSS allele(s), and therefore the presence of amylose in the starch granules, had a great influence on the physico-chemical properties of the starch suspensions.

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.