Physiological and gene expression response mechanisms of dehydration process in Cinnamomum cassia (L.) J. Presl seeds.

BACKGROUND: A critical factor in the storability of recalcitrant seeds is their moisture content (MC), but its effect on the viability of Cinnamomum cassia (L.) J. Presl (C. cassia) seeds is not fully understood. METHODS AND RESULTS: Measured the germination rate, starch and soluble sugar content, and transcriptome of 8 seed samples with different MC obtained by low-temperature drying method. It was found that the germination rate was significantly negatively correlated with MC. The lethal MC was around 15.6%. During the dehydration process, there was a significant increase in the content of soluble sugars and starch. Transcriptome analysis was performed on CK, W3, W6 showed a total of 62.78 Gb of clean data. Among the 30,228 Unigenes, 28,195 were successfully annotated. In the three comparative groups (CK and W3, CK and W6, W3 and W6), 6,842, 7,640, and 11,628 differentially expressed genes (DEGs) were obtained, respectively. These DEGs were found to be involved in a variety of metabolic pathways, including carbon metabolism, amino acid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, and nucleotide and amino sugar metabolism. A total of 1,416 common genes were identified among all three comparison groups. Furthermore, among all the DEGs, a total of 71 transcription factor families were identified, with the C2H2 transcription factor family having the highest number of genes. CONCLUSIONS: This ground-breaking study sheds light on the physiological response and gene expression profiles of C. cassia seeds after undergoing dehydration treatment, which will provide valuable insights for further research and understanding of this process.

Understanding the amylose biosynthesis and regulation mechanisms in Tartary buckwheat by the endosperm transcriptome.

Starch serves as a crucial energy source for both plants and humans, predominantly synthesized and stored in endosperms, tubers, rhizomes, and cotyledons. Given the significant role of amylose in determining the quality of starchy crops, optimizing its content has become a key objective in current crop breeding efforts. Tartary buckwheat, a dicotyledonous plant, notably accumulates high levels of amylose in its endosperm, surpassing common cereals like rice and maize. However, the mechanisms underlying amylose accumulation, distribution, and regulation in Tartary buckwheat remain unclear. Here, amylose content was determined across various tissues and organs of Tartary buckwheat, identifying with the endosperm as the primary site for its biosynthesis and accumulation. RNA sequencing analysis of endosperms from different developmental stages identified 35 genes potentially involved in starch biosynthesis, with 13 genes showing high endosperm-specific expression, suggesting crucial roles in starch biosynthesis. Additionally, the transcription factor FtNF-YB2, which was specifically highly expressed in the endosperm, was discovered to enhance amylose synthesis. Moreover, promoters with potential endosperm-specific activity were identified, advancing our understanding of amylose regulation. Additionally, this study also demonstrates that brassinosteroids (BR) positively influence amylose biosynthesis in Tartary buckwheat endosperm. These findings provide essential insights into the mechanisms of understanding amylose biosynthesis, accumulation and regulation in Tartary buckwheat, offering significant implications for future breeding strategies.

A comparison of the physicochemical properties, digestibility, and expression patterns of starch-related genes of two supersweet corn hybrids (F1) and their parents.

The quality difference of corn largely depends on parental selection. Herein, the structure, digestive characteristics, and expression patterns of starch-related genes of two supersweet maize hybrids and their parents were studied. The structural analysis revealed that the starch of supersweet corn is round or oval, and the particles are smaller compared to those of normal corn. Hybridization changed the grain morphology, crystal, and helical structure of starch. Parents had a significantly different influence on supersweet corn. Notably, hybridization improved the setback value and digestibility of Shantian1500F1 and Shantian2000F1 compared to that of the parents. ZmBEI, ZmPHOH, and ZmAGPL2 genes had a consistent high expression throughout the whole grain formation phase. The results of this study expand our understanding of the breeding of supersweet corn hybrids and the effect of parents on the new strand. These results provide a useful reference for further breeding and studies of supersweet corn for starch production in corn.

QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Deciphering the Transcriptional Regulatory Network Governing Starch and Storage Protein Biosynthesis in Wheat for Breeding Improvement.

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

ZmEREB92 plays a negative role in seed germination by regulating ethylene signaling and starch mobilization in maize.

Rapid and uniform seed germination is required for modern cropping system. Thus, it is important to optimize germination performance through breeding strategies in maize, in which identification for key regulators is needed. Here, we characterized an AP2/ERF transcription factor, ZmEREB92, as a negative regulator of seed germination in maize. Enhanced germination in ereb92 mutants is contributed by elevated ethylene signaling and starch degradation. Consistently, an ethylene signaling gene ZmEIL7 and an α-amylase gene ZmAMYa2 are identified as direct targets repressed by ZmEREB92. OsERF74, the rice ortholog of ZmEREB92, shows conserved function in negatively regulating seed germination in rice. Importantly, this orthologous gene pair is likely experienced convergently selection during maize and rice domestication. Besides, mutation of ZmEREB92 and OsERF74 both lead to enhanced germination under cold condition, suggesting their regulation on seed germination might be coupled with temperature sensitivity. Collectively, our findings uncovered the ZmEREB92-mediated regulatory mechanism of seed germination in maize and provide breeding targets for maize and rice to optimize seed germination performance towards changing climates.

Genome-Wide Investigation of BAM Gene Family in Annona atemoya: Evolution and Expression Network Profiles during Fruit Ripening.

ß-amylase proteins (BAM) are important to many aspects of physiological process such as starch degradation. However, little information was available about the BAM genes in Annona atemoya, an important tropical fruit. Seven BAM genes containing the conservative domain of glycoside hydrolase family 14 (PF01373) were identified with Annona atemoya genome, and these BAM genes can be divided into four groups. Subcellular localization analysis revealed that AaBAM3 and AaBAM9 were located in the chloroplast, and AaBAM1.2 was located in the cell membrane and the chloroplast. The AaBAMs belonging to Subfamily I contribute to starch degradation have the higher expression than those belonging to Subfamily II. The analysis of the expression showed that AaBAM3 may function in the whole fruit ripening process, and AaBAM1.2 may be important to starch degradation in other organs. Temperature and ethylene affect the expression of major AaBAM genes in Subfamily I during fruit ripening. These expressions and subcellular localization results indicating ß-amylase play an important role in starch degradation.

OsMADS1 Regulates Grain Quality, Gene Expressions, and Regulatory Networks of Starch and Storage Protein Metabolisms in Rice.

OsMADS1 plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated OsMADS1Olr allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1Olr-eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of OsMADS1 also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that OsMADS1 regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of OsMADS1 in regulating rice grain quality but also highlight the application potentials of OsMADS1 and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

TabHLH95-TaNF-YB1 module promotes grain starch synthesis in bread wheat.

Starch is the most abundant substance in wheat (Triticum aestivum L.) endosperm and provides the major carbohydrate energy for human daily life. Starch synthesis-related (SSR) genes are believed to be spatiotemporally specific, but their transcriptional regulation remains unclear in wheat. Here, we investigate the role of the basic helix-loop-helix (bHLH) transcription factor TabHLH95 in starch synthesis. TabHLH95 is preferentially expressed in the developing grains in wheat and encodes a nucleus localized protein without autoactivation activity. The Tabhlh95 knockout mutants display smaller grain size and less starch content than wild type, whereas overexpression of TabHLH95 enhances starch accumulation and significantly improves thousand grain weight. Transcriptome analysis reveals that the expression of multiple SSR genes is significantly reduced in the Tabhlh95 mutants. TabHLH95 binds to the promoters of ADP-glucose pyrophosphorylase large subunit 1 (AGPL1-1D/-1B), AGPL2-5D, and isoamylase (ISA1-7D) and enhances their transcription. Intriguingly, TabHLH95 interacts with the nuclear factor Y (NF-Y) family transcription factor TaNF-YB1, thereby synergistically regulating starch synthesis. These results suggest that the TabHLH95-TaNF-YB1 complex positively modulates starch synthesis and grain weight by regulating the expression of a subset of SSR genes, thus providing a good potential approach for genetic improvement of grain productivity in wheat.

Genetic diversity assessment and genome-wide association study reveal candidate genes associated with component traits in sweet potato (Ipomoea batatas (L.) Lam).

The Korean sweet potatoes were bred by various cultivars introduced from Japanese, American, Porto Rico, China, and Burundi. This issue enriched their genetic diversity but also resulted in a mixture of cultivars. For genotyping, we collected and sequenced 66 sweet potato germplasms from different localities around Korea, including 36 modern cultivars, 5 local cultivars, and 25 foreign cultivars. This identified 447.6 million trimmed reads and 324.8 million mapping reads and provided 39,424 single nucleotide polymorphisms (SNPs) markers. Phylogenetic clustering and population structure analysis distinctly classified these germplasms into 5 genetic groups, group 1, group 2, group 3, group 4, and group 5, containing 20, 15, 10, 7, and 14 accessions, respectively. Sixty-three significant SNPs were selected by genome-wide association for sugar composition-related traits (fructose, glucose, and total sugars), total starch, amylose content, and total carotenoid of the storage root. A total of 37 candidate genes encompassing these significant SNPs were identified, among which, 7 genes were annotated to involve in sugar and starch metabolism, including galactose metabolism (itf04g30630), starch and sucrose metabolism (itf03g13270, itf15g09320), carbohydrate metabolism (itf14g10250), carbohydrate and amino acid metabolism (itf12g19270), and amino sugar and nucleotide sugar metabolism (itf03g21950, itf15g04880). This results indicated that sugar and starch are important characteristics to determine the genetic diversity of sweet potatoes. These findings not only illustrate the importance of component traits to genotyping sweet potatoes but also explain an important reason resulting in genetic diversity of sweet potato.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco.

ß-amylase (BAM) plays an important role in plant development and response to abiotic stresses. In this study, 5 DoBAM members were identified in yam (Dioscorea opposita Thunb.). A novel ß-amylase gene BAM1, (named DoBAM1), was isolated from yam varieties Bikeqi and Dahechangyu. The open reading frame (ORF) of DoBAM1 is 2806 bp and encodes 543 amino acids. Subcellular localization analysis indicates that DoBAM1 localizes to the cell membrane and cytoplasm. In the yam variety Dahechangyu, the starch content, ß-amylase activity, and expression of DoBAM1 were characterized and found to all be higher than in Bikeqi. DoBAM1 overexpression in tobacco is shown to promote the accumulation of soluble sugar and chlorophyll content and to increase the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ß-amylase. Under cold treatment, we observed the induced upregulation of DoBAM1 and lower starch content and malondialdehyde (MDA) accumulation than in WT plants. In conclusion, these results demonstrate that DoBAM1 overexpression plays an advanced role in cold tolerance, at least in part by raising the levels of soluble sugars that are capable of acting as osmolytes or antioxidants.

Gene Expression in the Developing Seed of Wild and Domesticated Rice.

The composition and nutritional properties of rice are the product of the expression of genes in the developing seed. RNA-Seq was used to investigate the level of gene expression at different stages of seed development in domesticated rice (Oryza sativa ssp. japonica var. Nipponbare) and two Australian wild taxa from the primary gene pool of rice (Oryza meridionalis and Oryza rufipogon type taxa). Transcriptome profiling of all coding sequences in the genome revealed that genes were significantly differentially expressed at different stages of seed development in both wild and domesticated rice. Differentially expressed genes were associated with metabolism, transcriptional regulation, nucleic acid processing, and signal transduction with the highest number of being linked to protein synthesis and starch/sucrose metabolism. The level of gene expression associated with domestication traits, starch and sucrose metabolism, and seed storage proteins were highest at the early stage (5 days post anthesis (DPA)) to the middle stage (15 DPA) and declined late in seed development in both wild and domesticated rice. However, in contrast, black hull colour (Bh4) gene was significantly expressed throughout seed development. A substantial number of novel transcripts (38) corresponding to domestication genes, starch and sucrose metabolism, and seed storage proteins were identified. The patterns of gene expression revealed in this study define the timing of metabolic processes associated with seed development and may be used to explain differences in rice grain quality and nutritional value.

Natural Variation of Fatty Acid Desaturase Gene Affects Linolenic Acid Content and Starch Pasting Viscosity in Rice Grains.

Rice, as one of the main food crops, provides a vital source of dietary energy for over half the world's population. The OsFAD3 gene encodes fatty acid desaturase, catalyzing the conversion of linoleic acid (LA) to alpha-linolenic acid (ALA) in rice. However, the genetic characterization of OsFAD3 and its role in the conversion of LA to ALA remains elusive. Here, we validated the effects of two homologous genes, OsFAD3-1 and OsFAD3-2, on the ALA and LA/ALA ratio in rice grains using near-isogenic lines. Two major haplotypes of OsFAD3-1 are identified with different effects on the ALA and LA/ALA ratio in rice germplasm. High expression of OsFAD3-1 is associated with high ALA accumulation and eating quality of rice grains. Overexpression of OsFAD3-1 driven by a seed-specific promoter increases the ALA content up to 16-fold in the endosperm. A diagnostic marker is designed based on an 8-bp insertion/deletion in the OsFAD3-1 promoter, which can recognize OsFAD3-1 alleles in rice. These results indicate that OsFAD3-1 is a useful target gene in marker-assisted breeding programs to improve varieties with high ALA and appropriate LA/ALA ratio in brown rice.

Ectopic Expression of the Rice Grain-Size-Affecting Gene GS5 in Maize Affects Kernel Size by Regulating Endosperm Starch Synthesis.

Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and grain grouting rate. In this study, based on the GS5 transgenic maize obtained through transgenic technology with specific expression in the endosperm, molecular assays were performed on the transformed plants. Southern blotting results showed that the GS5 gene was integrated into the maize genome in a low copy number, and RT-PCR analysis showed that the exogenous GS5 gene was normally and highly expressed in maize. The agronomic traits of two successive generations showed that certain lines were significantly improved in yield-related traits, and the most significant changes were observed in the OE-34 line, where the kernel width increased significantly by 8.99% and 10.96%, the 100-kernel weight increased by 14.10% and 10.82%, and the ear weight increased by 13.96% and 15.71%, respectively; however, no significant differences were observed in the plant height, ear height, kernel length, kernel row number, or kernel number. In addition, the overexpression of the GS5 gene increased the grain grouting rate and affected starch synthesis in the rice grains. The kernels' starch content in OE-25, OE-34, and OE-57 increased by 10.30%, 7.39%, and 6.39%, respectively. Scanning electron microscopy was performed to observe changes in the starch granule size, and the starch granule diameter of the transgenic line(s) was significantly reduced. RT-PCR was performed to detect the expression levels of related genes in starch synthesis, and the expression of these genes was generally upregulated. It was speculated that the exogenous GS5 gene changed the size of the starch granules by regulating the expression of related genes in the starch synthesis pathway, thus increasing the starch content. The trans-GS5 gene was able to be stably expressed in the hybrids with the genetic backgrounds of the four materials, with significant increases in the kernel width, 100-kernel weight, and ear weight. In this study, the maize kernel size was significantly increased through the endosperm-specific expression of the rice GS5 gene, and good material for the functional analysis of the GS5 gene was created, which was of great importance in theory and application.

Novel Hina alleles created by genome editing increase grain hardness and reduce grain width in barley.

The hordoindolina genes (Hina and Hinb) are believed to play critical roles in barley (Hordeum vulgare L.) grain texture. In this study, we created novel alleles of the Hina gene using CRISPR/Cas9 (Clustered regularly inter spaced short palindromic repeat-associated protein, CRISPR-Cas) genome editing. Mutagenesis of single bases in these novel alleles led to loss of Hina protein function in edited lines. The grain hardness index of hina mutants was 95.5 on average, while that of the wild type was only 53.7, indicating successful conversion of soft barley into hard barley. Observation of cross-sectional grain structure using scanning electron microscopy revealed different adhesion levels between starch granules and protein matrix. Starch granules were loose and separated from the protein matrix in the wild type, but deeply trapped and tightly integrated with the protein matrix in hina02 mutants. In addition, the grain width and thousand-grain weight of the hina02 mutant were significantly lower than those of the wild type.

Physiological and omics analysis of maize inbred lines during late grain development.

BACKGROUND: There were significant differences in the change of moisture content and grain composition at the late stage of grain development among different maize varieties, but the regulation mechanism is not clear. OBJECTIVE: To explore the key genes causing the variation in physiological traits of two typical maize inbred lines in late grain development. METHODS: The grains at different development stages were selected as materials to determine the content of water, sucrose, starch and ABA. Transcriptomic and proteomic analysis of the materials were performed to screen relevant genes. RESULTS: The grain dehydration rate and the content of sucrose, starch and ABA were showed significant differences between two varieties in the late stage of grain development. The enrichment analysis of common differentially expressed genes (proteins) showed that most of the genes (proteins) were enriched in the extracellular region. The downregulated genes were mainly concentrated in carbohydrate metabolism and lipid metabolism, while the upregulated genes were mainly in response to stress. Furthermore, this study also identified many key candidate genes (dehydrin genes, pathogenesis-related genes, sucrose synthase and secondary metabolites related genes) related to late grain development of maize. CONCLUSIONS: The suggested genes related to late grain development of maize can be candidates for further functional study.

Comparative Study of Starch Phosphorylase Genes and Encoded Proteins in Various Monocots and Dicots with Emphasis on Maize.

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.