Shrunken Endosperm Mutant Research Articles

Loss of ADP-glucose transporter in barley sex1 mutant caused shrunken endosperm but with elevated protein and β-glucan content in whole meal

Grain shape and plumpness affect barley yield. Despite numerous studies on shrunken endosperm mutants in barley, their molecular mechanism and application potential in the food industry are largely unknown. Here, map-based cloning, co-segregation analyses, and allelic variant validation revealed that the loss of HORVU6Hr1G037950 encoding an ADP-glucose transporter caused the shrunken endosperm in sex1. Haplotype analysis suggested that hap4 in the promoter sequence was positively related to the hundred-grain weight showing a breeding potential. A pair of near-isogenic lines targeting HORVU6Hr1G037950 was produced and characterized to investigate molecular mechanisms that SEX1 regulates endosperm development. Results presented that the absence of the SEX1 gene led to the decrease of starch content and A-type granules size, the increase of β-glucan, protein, gelatinization temperature, soluble sugar content, amylopectin A chains, and B1 chains. Enzymatic activity, transcriptome and metabolome analyses revealed the loss of SEX1 results in an impaired ADP-glucose-to-starch conversion process, consequently leading to higher soluble sugar contents and lower starch accumulation, thereby inducing a shrunken-endosperm phenotype in sex1. Taken together, this study provides new insights into barley grain development, and the elevated protein and β-glucan contents of the whole meal in sex1 imply its promising application in the food industry.

Identification and characterization of mRNAs and lncRNAs of a barley shrunken endosperm mutant using RNA-seq.

Barley shrunken endosperm mutants have been extensively reported. However, knowledge of the underlying molecular mechanisms of these mutants remains limited. Here, a pair of near isogenic lines (normal endosperm: Bowman and shrunken endosperm: sex1) was subjected to transcriptome analysis to identify mRNAs and lncRNAs related to endosperm development to further dissect its mechanism of molecular regulation. A total of 2123 (1140 up- and 983 down-regulated) unique differentially expressed genes (DEGs) were detected. Functional analyses showed that these DEGs were mainly involved in starch and sucrose metabolism, biosynthesis of secondary metabolites, and plant hormone signal transduction. A total of 343 unique target genes were identified for 57 differentially expressed lncRNAs (DE lncRNAs). These DE lncRNAs were mainly involved in glycerophospholipid metabolism, starch and sucrose metabolism, hormone signal transduction, and stress response. In addition, key lncRNAs were identified by constructing a co-expression network of the target genes of DE lncRNAs. Transcriptome results suggested that mRNA and lncRNA played a critical role in endosperm development. The shrunken endosperm in barley seems to be closely related to plant hormone signal transduction, starch and sucrose metabolism, and cell apoptosis. This study provides a foundation for fine mapping, elucidates the molecular mechanism of shrunken endosperm mutants, and also provides a reference for further studies of lncRNAs during the grain development of plants.

Barley lys3 mutants are unique amongst shrunken-endosperm mutants in having abnormally large embryos

Many shrunken endosperm mutants of barley (Hordeum vulgare L.) have been described and several of these are known to have lesions in starch biosynthesis genes. Here we confirm that one type of shrunken endosperm mutant, lys3 (so called because it was first identified as a high-lysine mutant) has an additional phenotype: as well as shrunken endosperm it also has enlarged embryos. The lys3 embryos have a dry weight that is 50–150% larger than normal. Observations of developing lys3 embryos suggest that they undergo a form of premature germination and the mature lys3 grains show reduced dormancy. In many respects, the phenotype of barley lys3 is similar to that of rice GIANT EMBRYO mutants (affected in the OsGE gene). However, the barley orthologue of OsGE is located on a different chromosome from Lys3. Together these results suggest that the gene underlying Lys3 is unlikely to encode a starch biosynthesis protein but rather a protein influencing grain development.

Spatio-temporal dynamics of fructan metabolism in developing barley grains.

Barley (Hordeum vulgare) grain development follows a series of defined morphological and physiological stages and depends on the supply of assimilates (mainly sucrose) from the mother plant. Here, spatio-temporal patterns of sugar distributions were investigated by mass spectrometric imaging, targeted metabolite analyses, and transcript profiling of microdissected grain tissues. Distinct spatio-temporal sugar balances were observed, which may relate to differentiation and grain filling processes. Notably, various types of oligofructans showed specific distribution patterns. Levan- and graminan-type oligofructans were synthesized in the cellularized endosperm prior to the commencement of starch biosynthesis, while during the storage phase, inulin-type oligofructans accumulated to a high concentration in and around the nascent endosperm cavity. In the shrunken endosperm mutant seg8, with a decreased sucrose flux toward the endosperm, fructan accumulation was impaired. The tight partitioning of oligofructan biosynthesis hints at distinct functions of the various fructan types in the young endosperm prior to starch accumulation and in the endosperm transfer cells that accomplish the assimilate supply toward the endosperm at the storage phase.

Gibberellin-to-abscisic acid balances govern development and differentiation of the nucellar projection of barley grains.

In cereal grains, the maternal nucellar projection (NP) constitutes the link to the filial organs, forming a transfer path for assimilates and signals towards the endosperm. At transition to the storage phase, the NP of barley (Hordeum vulgare) undergoes dynamic and regulated differentiation forming a characteristic pattern of proliferating, elongating, and disintegrating cells. Immunolocalization revealed that abscisic acid (ABA) is abundant in early non-elongated but not in differentiated NP cells. In the maternally affected shrunken-endosperm mutant seg8, NP cells did not elongate and ABA remained abundant. The amounts of the bioactive forms of gibberellins (GAs) as well as their biosynthetic precursors were strongly and transiently increased in wild-type caryopses during the transition and early storage phases. In seg8, this increase was delayed and less pronounced together with deregulated gene expression of specific ABA and GA biosynthetic genes. We concluded that differentiation of the barley NP is driven by a distinct and specific shift from lower to higher GA:ABA ratios and that the spatial-temporal change of GA:ABA balances is required to form the differentiation gradient, which is a prerequisite for ordered transfer processes through the NP. Deregulated ABA:GA balances in seg8 impair the differentiation of the NP and potentially compromise transfer of signals and assimilates, resulting in aberrant endosperm growth. These results highlight the impact of hormonal balances on the proper release of assimilates from maternal to filial organs and provide new insights into maternal effects on endosperm differentiation and growth of barley grains.

Characterization of shrunken endosperm mutants in barley

Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1–seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27days after anthesis (DAA) after reaching the peak at 15–21DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

De-regulation of abscisic acid contents causes abnormal endosperm development in the barley mutant seg8

Grain development of the maternal effect shrunken endosperm mutant seg8 was analysed by comprehensive molecular, biochemical and histological methods. The most obvious finding was de-regulation of ABA levels, which were lower compared to wild-type during the pre-storage phase but higher during the transition from cell division/differentiation to accumulation of storage products. Ploidy levels and ABA amounts were inversely correlated in the developing endosperms of both mutant and wild-type, suggesting an influence of ABA on cell-cycle regulation. The low ABA levels found in seg8 grains between anthesis and beginning endosperm cellularization may result from a gene dosage effect in the syncytial endosperm that causes impaired transfer of ABA synthesized in vegetative tissues into filial grain parts. Increased ABA levels during the transition phase are accompanied by higher chlorophyll and carotenoid/xanthophyll contents. The data suggest a disturbed ABA-releasing biosynthetic pathway. This is indicated by up-regulation of expression of the geranylgeranyl reductase (GGR) gene, which may be induced by ABA deficiency during the pre-storage phase. Abnormal cellularization/differentiation of the developing seg8 endosperm and reduced accumulation of starch are phenotypic characteristics that reflect these disturbances. The present study did not reveal the primary gene defect causing the seg8 phenotype, but presents new insights into the maternal/filial relationships regulating barley endosperm development.

PENGARUH BEBERAPA FITOHORMON PADA PEMBENTUKAN MUTAN BARLEY (Hordeum Vulgare) DENGAN FENOTIP BIJI ABNORMAL

Seg8 is the shrunken endosperm mutant occured naturally in barley (Hordeum vulgare). This recessive mutant does not express xenia which indicates that the endosperm phenotypes depend on the genotype of the diploid maternal parents. This mutant provides an opportunity to analyze factors produced by the female parent that affect the seed development process. One of the factors affecting seed development and growth is phytohormones. In vitro spike culture system adopted from wheat spike culture system was used to investigate the involvement of phytohormones in the grain filling process. Phytohormones were applied during 15 days period of culture. Phytohormones concentrations used were as follows abscisic acid 10–4 M, 5x10–5 M, 10–6M, 5x10–7M and 10–7M. Gibberelic acid concentrations were 5x10–3 M, 5x10–4 M, 5x10–5 M, 2.5x10–5M, 10-5M and 2x10–6M. Cytokinin concentrations were 2x10–6 M, 2x10–7 M and 2x10–8 M. In this experiment, 2 days before anthesis is the best stage to start the spike culture period and 15 days period of culture is sufficient to observe the grain filling process and emergence of the mutant phenotype. The phytohormones treatments as well as their respective concentrations were not able to recover the normal phenotype. Abscisic acid treatment at 5x10-7 M were able to increase the mutant grain dry weight significantly compared to untreated culture but the normal phenotype was not recovered. This indicates that lack of phytohormones was not the maternal factor affecting the seed development process in this particular mutant.

Endosperm differentiation in barley wild‐type and sex mutants

The cereal endosperm is a storage organ consisting of the central starchy endosperm surrounded by the aleurone layer. In barley, endosperm development is subdivisible into four main stages, i.e. the syncytial (I), the cellularization (II), the differentiation (III) and the maturation stage (IV). During stage I, a multinucleate syncytium is formed, which in stage II develops into the undifferentiated cellular endosperm. During stage III the cells of the endosperm differentiate into two types of aleurone cells (peripheral and modified) and three different starchy endosperm cell types (irregular, prismatic and subaleurone).To elucidate the ontogenetic relationship between the endosperm tissues, the phenotypes of sex (shrunken endosperm mutants expressing xenia) mutant endosperms were studied. These mutants can be classified into two groups, i.e. those in which development is arrested at one of the four wild‐type stages described above, and those with abnormal development with new organizational patterns in the endosperm or with novel cell types. Based on these studies, it is suggested that the two endosperm halves represent cell lines derived from the two daughter nuclei of the primary endosperm nucleus, and that the prismatic starchy endosperm cells arise from a peripheral endosperm meristematic activity during stage III. Finally, a model for the main molecular events underlying the morphogenetic processes is discussed.

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley (Hordeum vulgare L. cv. Bomi‐like), shx, which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP‐glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule‐bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP‐glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch‐bound starch synthase activities are normal, but ADP‐glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer‐independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer‐dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch‐synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary‐site of the mutation in shx.

A novel shrunken endosperm mutant of barley

A novel shrunken endosperm mutant of barley

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 3∶1 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.

Growth Characteristics, Grain Filling, and Assimilate Transport in a Shrunken Endosperm Mutant of Barley

The reported inheritance pattern of the seg1 shrunken endosperm mutant of barley (Hordeum vulgare L. cv Betzes) suggests that some defective process in the maternal plant tissues, and not in the endosperm, prevents normal grain filling in the mutant. To identify the physiological mechanism of the mutation, we compared growth, carbon exchange, and assimilate transport of Betzes and seg1 plants. Betzes and seg1 plants did not differ in mean relative growth rate, mean net assimilation rate, or carbon exchange rate. The rate and duration of grain growth of seg1 was lower than Betzes on intact plants and on detached, cultured spikes. Increasing the supply of sucrose in culture media up to 300 mm sucrose did not eliminate differences between normal and mutant grain growth. Translocation of (14)C-labeled assimilates into seg1 grains ceased by 21 days after anthesis, and assimilates were diverted to lower plant parts. In contrast, assimilates were still entering Betzes grains at 29 days after anthesis. Evidence suggests that some maternal spike or grain tissue is affected by the mutation after the onset of grain filling. Identification of the specific seg1 defect may provide information about the cessation of normal grain filling.

Lysine and Protein Characterization of Induced Shrunken Endosperm Mutants of Barley1

‘Bomi’, Risø mutants 7, 8, 9, 13, and 1508 and ‘Carlsberg II’, Risø mutants 29, 56 and 86, induced high lysine shrunken endosperm and/or low kernel weight mutants of barley (Hordeum vulgare L.), were compared to their respective normal isotypes for kernel weight, protein, and lysine contents in several varied environments in Montana and Arizona. All mutants were lower in kernel weight and higher in lysine content, and six of the mutants were higher in protein content, with a relatively wide range of mutant‐normal differences for each character. Most of the mutants should maintain a positive mutant‐normal difference in lysine percentage if normal kernel weights were achieved through breeding efforts. Both modes of mutagenesis, induced and spontaneous, and two methods of selection, chemical (for high lysine) and visual (for shrunken endosperm), have produced a similar spectrum of high lysine, shrunken endosperm mutants.

Lysine and Protein Characterization of Spontaneous Shrunken Endosperm Mutants of Barley1

Compana sex1, seg3 and seg4, Betzes seg1 and seg2, Ingrid seg6 and seg7, Glacier seg5, High Amylose Glacier amol and Hiproly lysl, spontaneous shrunken endosperm or low kernel weight mutants of barley (Hordeum vulgare L.), were compared to their normal isotypes for kernel weight, protein, and lysine contents. All mutants were lower in kernel weight and most were higher in protein and lysine contents than respective normal isotypes indicating that at least seven new mutants should be added to the list of known “high lysine” mutants of barley, and confirming the observation in barley that low seed weight and high lysine are closely associated. Mutants sex1, seg4, amol and perhaps Hiproly show the most promise of overcoming the detrimental high lysine‐low kernel weight association in a breeding program.

Shrunken Endosperm Mutants in Barley1

AbstractSix spontaneous shrunken endosperm mutants in barley (Hordeum vulgare L.) were identified and described. Five of the mutants (se1 through se5) were inherited as maternal plant monofactorial recessives and one mutant se6 was inherited as a monofactorial recessive expressing xenia. The mutants varied in fertility, seed weight, and seive size assortment. Four mutants were located on chromosome one, one on chromosome three, and one on chromosome six, by using translocation breakpoints as markers. Mutants similar to se1 through se5 could be used as males in commerical hybrid seed production. Mutants similar to se6 could be used as preflowering selective genes for the female stock for hybrid seed production.