Identification and analysis of nine new flo2 allelic mutants in rice.

FLO2 is involved in grain development and storage substance synthesis in rice, and therefore can regulate grain size and quality. In this study, we identified 4 new flo2 allelic mutants with nonsense and frameshift mutation in the exon of 6, 10, 11 and 21 and 5 new flo2 allelic mutants with alternative splicing and frameshift mutation at the splicing site of intron 13, 14, 16 and 17. Compared with wild-type rice, the outer endosperm of flo2 mutants was transparent, and the inner endosperm was floury. Different mutation sites and types of FLO2 significantly decreased kernel width, thickness and weight to some extent. The contents of storage protein, starch, amylose and amylopectin showed significant decrease at different levels among 9 flo2 mutants. The expressions of most storage protein synthesis genes and starch synthesis-related genes were significantly down-regulated, and exhibited different ranges of variation among different flo2 mutants. This study could add helpful information for the roles of flo2 alleles in rice quality regulation and provide abundant germplasm resources for rice quality breeding.

Structural, Thermal, Pasting and Digestion Properties of Starches from Developing Root Tubers of Sweet Potato.

Three sweet potato varieties with white-, yellow- and purple-fleshed root tubers were harvested at 100, 120, 140 and 160 days after planting (DAP). Their starch structural, thermal, pasting and digestion properties were measured to reveal the influences of harvesting dates on the physicochemical properties of sweet potato root tuber starch. Though starches from different varieties displayed some differences in physicochemical properties due to their different genetic backgrounds, they were influenced by harvesting date in similar ways. Starches isolated from root tubers at 100 and 160 DAP exhibited lower granule sizes than those at 120 and 140 DAP. The amylose content was higher in root tubers at 100 and 120 DAP than at 140 and 160 DAP. Starches from root tubers at 100 DAP exhibited CA-type X-ray diffraction patterns, and then the B-type crystallinity gradually increased at later harvesting dates. The different harvesting dates had no significant effects on the short-ranged ordered structure and lamellar thickness of starch, but the lamellar peak intensity decreased significantly at later harvesting dates. Starch had a lower gelatinization temperature and a wider gelatinization temperature range in root tubers at 140 and 160 DAP than at 100 and 120 DAP. The higher peak viscosity and lower pasting temperature were associated with the late harvesting date. The digestion of starch had slight differences among root tubers at different harvesting dates. The harvesting dates of root tubers played more important roles in starch properties than the variety. This study would be helpful for breeders, farmers and sweet potato starch users.

Physicochemical properties of starches from sweet potato root tubers grown in natural high and low temperature soils.

Three sweet potato varieties grew in natural high temperature (HT) and low temperature (LT) field soils. Their starch physicochemical properties were affected similarly by HT and LT soils. Compared with LT soil, HT soil induced the increases of granule size D[4,3] from 18.0-18.7 to 19.9-21.8 µm and amylopectin average branch-chain length from 21.9-23.1 to 24.1-24.7 DP. Starches from root tubers grown in HT and LT soils exhibited CA- and CC-type XRD pattern, respectively. Starches from root tubers grown in HT soil exhibited stronger lamellar peak intensities (366.8-432.0) and higher gelatinization peak temperature (72.0-76.8 °C) than those (176.2-260.5, 56.4-63.4 °C) in LT soil. Native starches from root tubers grown in LT soil were hydrolyzed more easily (hydrolysis rate coefficient 0.227-0.282 h-1) by amylase than those (0.120-0.163 h-1) in HT soil. The principal component analysis exhibited that starches from root tubers grown in HT and LT soils had significantly different physicochemical properties.

Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis-related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25-OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis-related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis-related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis-related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis-related genes and normal starch accumulation.

G1 Interacts with OsMADS1 to Regulate the Development of the Sterile Lemma in Rice.

Flower development, as the basis for plant seed development, is principally conserved in angiosperms. At present, a number of genes regulating flower organ differentiation have been identified, and an ABCDE model has also been proposed. In contrast, the mechanism that regulates the development of the sterile lemma remains unclear. In this study, we identified and characterized a rice floral organ mutant, M15, in which the sterile lemma transformed into a lemma-like organ. Positional cloning combined with a complementary experiment demonstrated that the mutant phenotype was restored by LONG STERILE LEMMA1/(G1). G1 was expressed constitutively in various tissues, with the highest expression levels detected in the sterile lemma and young panicle. G1 is a nucleus-localized protein and functions as a homomer. Biochemical assays showed that G1 physically interacted with OsMADS1 both in vitro and in vivo. Interestingly, the expression of G1 in M15 decreased, while the expression level of OsMADS1 increased compared with the wild type. We demonstrate that G1 plays a key role in sterile lemma development through cooperating with OsMADS1. The above results have implications for further research on the molecular mechanisms underlying flower development and may have potential applications in crop improvement strategies.

Staining Starch with Iodine Solution.

Starch is important material in plant tissues, especially for storage tissues. Starches from different plant resources or tissues vary in morphology, content, and physicochemical properties. Starch and iodine can bind specifically to present the shapes and sizes of starch granules in plant tissues. Here, we describe some methods for staining starch in leaf, pollen grain, and starchy seeds with iodine solution. In addition, the isolated starch can also be stained with iodine solution to exhibit its shape and size.

Effects of growth temperature on multi-scale structure of root tuber starch in sweet potato.

Sweet potato was planted at three soil and air temperatures (21, 25 and 28 °C) with the same humidity and light time/intensity. Root tuber starches were isolated, and their multi-scale structures were investigated to reveal the effects of growth temperature on starch properties. Growth temperature did not change the morphology and amylose content of starch, but markedly increased the size of starch from volume-weighted mean diameter 12.2 µm to 17.0 µm. Starch grown at high growth temperature exhibited less A branch-chains and lower branching degree of amylopectin and more B2 and B3+ branch-chains of amylopectin than at low growth temperature. With increasing growth temperature, starch changed from CC-type to CA-type, its relative crystallinity and lamellar peak intensity increased, and the thickness of crystalline and amorphous lamellae did not significantly change. Starch grown at high growth temperature exhibited significantly higher gelatinization temperature than at low growth temperature, but had similar gelatinization enthalpy.

Relationships between X-ray Diffraction Peaks, Molecular Components, and Heat Properties of C-Type Starches from Different Sweet Potato Varieties.

C-type starches with different proportions of A- and B-type crystallinities have different intensities and crystallinities of X-ray diffraction peaks. In this study, the intensities and crystallinities of X-ray diffraction peaks, molecular components and heat properties of C-type starches were investigated in seven sweet potato varieties, and their relationships were analyzed. The intensity and crystallinity of a diffraction peak at 5.6° were significantly positively correlated to the DP6-12 branch-chains of amylopectin and significantly negatively correlated to the true amylose content (TAC) determined by concanavalin A precipitation, gelatinization temperature, gelatinization enthalpy, water solubility at 95 °C, and pasting temperature. The intensity of diffraction peaks at 15° and 23° were significantly positively correlated to the gelatinization temperature and pasting temperature and significantly negatively correlated to the pasting peak viscosity. The significantly positive relationships were detected between the crystallinity of a diffraction peak at 15° and the DP13-24 branch-chains of amylopectin, gelatinization conclusion temperature and water solubility, between the crystallinity of diffraction peak at 17-18° and the TAC, gelatinization onset temperature, water solubility and pasting temperature, between the crystallinity of a diffraction peak at 23° and the gelatinization conclusion temperature and pasting peak time, and between the total crystallinity and the TAC, gelatinization conclusion temperature, water solubility and pasting temperature. The score plot of principle component analysis showed that the molecular components and heat property parameters could differentiate the C-type starches and agreed with their characteristics of X-ray diffraction peaks. This study provides some references for the utilizations of C-type starches.

Sizes, Components, Crystalline Structure, and Thermal Properties of Starches from Sweet Potato Varieties Originating from Different Countries.

Sweet potato is a root tuber crop and an important starch source. There are hundreds of sweet potato varieties planted widely in the world. Starches from varieties with different genotype types and originating from different countries have not been compared for their physicochemical properties. In the research, starches from 44 sweet potato varieties originating from 15 countries but planted in the same growing conditions were investigated for their physicochemical properties to reveal the similarities and differences in varieties. The results showed that the 44 starches had granule size (D[4,3]) from 8.01 to 15.30 µm. Starches had different iodine absorption properties with OD680 from 0.259 to 0.382 and OD620/550 from 1.142 to 1.237. The 44 starches had apparent amylose content from 19.2% to 29.2% and true amylose content from 14.2% to 20.2%. The starches exhibited A-, CA-, CC-, or CB-type X-ray diffraction patterns. The thermograms of 44 starches exhibited one-, two-, or three-peak curves, leading to a significantly different gelatinization temperature range from 13.1 to 29.2 °C. The significantly different starch properties divide the 44 sweet potato varieties into different groups due to their different genotype backgrounds. The research offers references for the utilization of sweet potato germplasm.

Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice.

Bi-allelic mutant lines induced by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems are important genetic materials. It is very important to establish a rapid and cheap method in identifying homozygous mutant plants from offspring segregation populations of bi-allelic mutant lines. In this study, the offspring genotypes of rice bi-allelic starch branching enzyme IIb mutant lines were identified using the allele specific PCR (AS-PCR) method. The target sequences of two alleles were aligned from their 5' to 3' ends, and the first different bases were used as the 3' ends of mismatch primers. Another mismatched base was introduced at the third nucleotide from the 3' end of mismatch primer. The PCR reaction mixture and amplification program were optimized according to the differences of mutation target sequence and mismatch primers. The offspring plant genotypes of bi-allelic mutant lines could be accurately identified using the amplified DNA fragments by agarose gel electrophoresis. This study could provide a method reference for the rapid screening of homozygous mutant plants from offspring segregation population of heterozygous and bi-allelic mutant lines.

The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

Effects of Variety and Growing Location on Physicochemical Properties of Starch from Sweet Potato Root Tuber.

Three sweet potato varieties with purple-, yellow-, and white-fleshed root tubers were planted in four growing locations. Starches were isolated from their root tubers, their physicochemical properties (size, iodine absorption, amylose content, crystalline structure, ordered degree, lamellar thickness, swelling power, water solubility, and pasting, thermal and digestion properties) were determined to investigate the effects of variety and growing location on starch properties in sweet potato. The results showed that granule size (D[4,3]) ranged from 12.1 to 18.2 µm, the iodine absorption parameters varied from 0.260 to 0.361 for OD620, from 0.243 to 0.326 for OD680 and from 1.128 to 1.252 for OD620/550, and amylose content varied from 16.4% to 21.2% among starches from three varieties and four growing locations. Starches exhibited C-type X-ray diffraction patterns, and had ordered degrees from 0.634 to 0.726 and lamellar thicknesses from 9.72 to 10.21 nm. Starches had significantly different swelling powers, water solubilities, pasting viscosities, and thermal properties. Native starches had rapidly digestible starch (RDS) from 2.2% to 10.9% and resistant starch (RS) from 58.2% to 89.1%, and gelatinized starches had RDS from 70.5% to 81.4% and RS from 10.8% to 23.3%. Two-way ANOVA analysis showed that starch physicochemical properties were affected significantly by variety, growing location, and their interaction in sweet potato.

Characterization of underutilized root starches from eight varieties of ramie (Boehmeria nivea) grown in China.

Ramie root is an underutilized starch source. In this study, eight ramie varieties were investigated for starch properties. Starch content ranged from 18.6% to 50.1% in dry root. Starches from different varieties showed similar morphology including ellipsoidal, spherical and truncated granules with size D[4,3] from 10.1 to 14.1 µm. Starch had amylose content from 20.8% to 28.5%. All ramie varieties had B-type starches with relative crystallinity from 24.8% to 27.1%, ordered degree from 0.724 to 0.897 and lamellar thickness from 9.1 to 9.6 nm. Starches had gelatinization peak temperature from 70.5 to 73.8 °C and enthalpy from 14.9 to 15.8 J/g. Starches had swelling power and water solubility from 27.9 to 31.9 g/g and from 11.7% to 15.5%, respectively, at 95 °C, and exhibited different pasting properties with breakdown viscosity from 36 to 377 mPa s and setback viscosities from 1295 to 1863 mPa s. Starch pastes exhibited pseudoplastic behavior and different rheological properties. Native, gelatinized and retrograded starches had resistant starch from 81.7% to 83.9%, from 1.7% to 5.1% and from 5.6% to 13.3%, respectively. The eight varieties were divided into 3 groups according to starch properties. This study is helpful for selecting suitable ramie variety as starch source.

Physicochemical properties of a new starch from ramie (Boehmeria nivea) root.

A new starch was isolated from ramie root, and its physicochemical properties were investigated. Ramie dry root contained 45.9% starch. Starch had truncated, ellipsoidal, and spherical granule shapes with size from 7 to 30 µm and D[4,3] about 14.1 µm. Starch contained 38.9% apparent amylose content and 22.4% true amylose content, exhibited B-type crystallinity, and had 26.6% relative crystallinity, 0.82 ordered degree, and 9.2 nm lamellar thickness. Starch had 71.8 °C gelatinization peak temperature and 15.6 J/g gelatinization enthalpy, and exhibited 31.4 g/g swelling power and 17.1% water solubility at 95 °C. Starch had peak, hot, breakdown, final, and setback viscosities at 3048, 2768, 279, 4165, and 1397 mPa s, respectively, and showed peak time at 4.36 min and pasting temperature at 75.0 °C. The native, gelatinized, and retrograded starches contained 15.1%, 94.0%, and 86.5% rapidly digestible starch and 83.3%, 4.0%, and 10.7% resistant starch, respectively. Compared with potato and rice starches, ramie starch was somewhat similar to potato starch but significantly different from rice starch in starch component, crystalline structure, and functional properties. Therefore, ramie starch exhibited the potential to be used as a thickening agent, resistant-digesting food additive, and alternative to potato starch in food and nonfood industries.

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications.

The morphology, size and quantity of cells, starch granules and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

The NAC Transcription Factors OsNAC20 and OsNAC26 Regulate Starch and Storage Protein Synthesis.

Starch and storage proteins determine the weight and quality of cereal grains. Synthesis of these two grain components has been comprehensively investigated, but the transcription factors responsible for their regulation remain largely unknown. In this study, we investigated the roles of NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20, and OsNAC26 in starch and storage protein synthesis in rice (Oryza sativa) endosperm. OsNAC20 and OsNAC26 showed high levels of amino acid sequence similarity. Both were localized in the aleurone layer, starchy endosperm, and embryo. Mutation of OsNAC20 or OsNAC26 alone had no effect on the grain, while the osnac20/26 double mutant had significantly decreased starch and storage protein content. OsNAC20 and OsNAC26 alone could directly transactivate the expression of starch synthaseI (SSI), pullulanase (Pul), glutelin A1 (GluA1), glutelin B4/5 (GluB4/5), α-globulin, and 16 kD prolamin and indirectly influenced plastidial disproportionating enzyme1 (DPE1) expression to regulate starch and storage protein synthesis. Although they could also bind to the promoters of ADP-Glc pyrophosphorylase small subunit 2b (AGPS2b), ADP-Glc pyrophosphorylase large subunit 2 (AGPL2), and starch branching enzymeI (SBEI), and the expression of the three genes was largely decreased in the osnac20/26 mutant, ADP-Glc pyrophosphorylase and starch branching enzyme activities were unchanged in this double mutant. In addition, OsNAC20 and OsNAC26 are main regulators of Pul, GluB4, α-globulin, and 16 kD prolamin In conclusion, OsNAC20 and OsNAC26 play an essential and redundant role in the regulation of starch and storage protein synthesis.

Morphology, structure, properties and applications of starch ghost: A review.

Starch ghost, an insoluble structure of gelatinized starch, plays an important role in the applications of starch. In this review, we summarized the preparation, morphology, structure, properties and applications of starch ghost. The preparation steps of starch ghost include gelatinization, purification and preservation, and many factors influence the yield of starch ghost. The morphology and content of starch ghost can be influenced by many factors like starch resource and amylose content. Ghosts from non-waxy starches are composed of amylopectin with long branch-chains and amylose. These molecules cross-link to each other to reinforce the structure, and tend to form B-type double helix in ghosts from high-amylose starches. Some surface proteins that bind tightly to starch granules are also present in starch ghost. Protein and lipid are thought to have limited effects on the structural stability, but they make a big difference in the morphology of starch ghost. Starch ghost shows a different resistance to amylase among various starches, but it can be further digested under the high shear force. The mechanical, enzymatic hydrolysis and electrochemical properties of starch ghost make it widely used as emulsifier, stabilizer, thickener and starch-based films or gels in food and non-food processing industries.

Effects of nitrogen level on structural and functional properties of starches from different colored-fleshed root tubers of sweet potato.

The nitrogen (N) influences the growth of sweet potato. However, it is unclear whether the different levels of N can affect starch physicochemical properties. In this study, 9 different colored-fleshed sweet potato varieties were planted in the same field with additional N fertilizer treatment of 0, 15 and 30 kg/ha. The physicochemical properties of starches from root tubers were measured. With increasing N level, the amylose content decreased in yellow-fleshed variety Sushu 16 and increased in white-fleshed variety Sushu 29 and purple-fleshed varieties Ningzishu 1 and 4, but did not significantly change in other varieties. The starch size decreased in purple-fleshed variety Ningzishu 1 and white-fleshed varieties Sushu 28 and Sushu 29 with increasing N treatment, but first increased then decreased in yellow-fleshed variety Sushu 16 and first decreased then increased in white-fleshed variety Sushu 24 and yellow-fleshed variety Sushu 25. The different levels of N treatment had no influence on protein content, crystalline structure, and gelatinization enthalpy of starch. The effects of N treatment on gelatinization temperatures and pasting viscosities of starches were determined by varieties and genotype backgrounds of sweet potato. The PLSR and PLS-DA were also carried out based on structural, thermal, and pasting parameters of starches.

The Kernel Size-Related Quantitative Trait Locus qKW9 Encodes a Pentatricopeptide Repeat Protein That Aaffects Photosynthesis and Grain Filling.

In maize (Zea mays), kernel weight is an important component of yield that has been selected during domestication. Many genes associated with kernel weight have been identified through mutant analysis. Most are involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, few quantitative trait loci (QTLs) underlying quantitative variation in kernel weight have been cloned. Here, we characterize a QTL, qKW9, associated with maize kernel weight. This QTL encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of ndhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qkw9 background, C-to-U editing of ndhB was abolished, and photosynthesis was reduced, resulting in less maternal photosynthate available for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis for maize grain yield production.

The defective effect of starch branching enzyme IIb from weak to strong induces the formation of biphasic starch granules in amylose-extender maize endosperm.

KEYMESSAGE: Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.