A pan-grass transcriptome reveals patterns of cellular divergence in crops.

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits1. Key traits that distinguish these species are mediated by specialized cell types2. Here we compare the transcriptomes of root cells in three grass species-Zea mays, Sorghum bicolor and Setaria viridis. We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others-driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.

Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

Peptide Mapping, In Silico and In Vivo Analysis of Allergenic Sorghum Profilin Peptides.

BACKGROUND AND OBJECTIVES: Nearly 20-30% of the world's population suffers from allergic rhinitis, among them 15% are progressing to asthma conditions. Sorghum bicolor profilin (Sorb PF), one of the panallergens, was identified, but the allergen specificity is not yet characterized. MATERIALS AND METHODS: To map the antigenic determinants responsible for IgE binding, the present study is focused on in silico modeling, simulation of Sorb PF and docking of the Sorb PF peptides (PF1-6) against IgG and IgE, followed by in vivo evaluation of the peptides for its allergenicity in mice. RESULTS: Peptide PF3 and PF4 displayed high docking G-scores (-9.05) against IgE only. The mice sensitized with PF3 peptide showed increased levels of IL5, IL12, TNF-alpha, and GMCSF when compared to other peptides and controls, signifying a strong, Th2-based response. Concurrently, the Th1 pathway was inhibited by low levels of cytokine IL2, IFN-γ, and IL-10 justifying the role of PF3 in allergenic IgE response. CONCLUSIONS: Based on the results of overlapping peptides PF3 and PF4, the N-terminal part of the PF3 peptide (TGQALVI) plays a crucial role in allergenic response of Sorghum profilin.

Interplay between silica deposition and viability during the life span of sorghum silica cells.

Silica cells are specialized epidermal cells found on both surfaces of grass leaves, with almost the entire lumen filled with solid silica. The mechanism precipitating silicic acid into silica is not known. Here we investigate this process in sorghum (Sorghum bicolor) leaves. Using fluorescent confocal microscopy, we followed silica cells' ontogeny, aiming to understand the fate of vacuoles and nuclei. Correlating the confocal and scanning electron microscopy, we timed the initiation of silica deposition in relation to cell's viability. Contrary to earlier reports, silica cells did not lose their nucleus before silica deposition. Vacuoles in silica cells did not concentrate silicic acid. Instead, postmaturation silicification initiated at the cell periphery in live cells. Less than 1% silica cells showed characteristics of programmed cell death in the cell maturation zone. In fully elongated mature leaves, 2.4% of silica cells were nonsilicified and 1.6% were partially silicified. Silica deposition occurs in the paramural space of live silica cells. The mineral does not kill the cells. Instead, silica cells are genetically programmed to undergo cell death independent of silicification. Fully silicified cells seem to have nonsilicified voids containing membrane remains after the completion of the cell death processes.

Mechanism of silica deposition in sorghum silica cells.

Grasses take up silicic acid from soil and deposit it in their leaves as solid silica. This mineral, comprising 1-10% of the grass dry weight, improves plants' tolerance to various stresses. The mechanisms promoting stress tolerance are mostly unknown, and even the mineralization process is poorly understood. To study leaf mineralization in sorghum (Sorghum bicolor), we followed silica deposition in epidermal silica cells by in situ charring and air-scanning electron microscopy. Our findings were correlated to the viability of silica cells tested by fluorescein diacetate staining. We compared our results to a sorghum mutant defective in root uptake of silicic acid. We showed that the leaf silicification in these plants is intact by detecting normal mineralization in leaves exposed to silicic acid. Silica cells were viable while condensing silicic acid into silica. The controlled mineral deposition was independent of water evapotranspiration. Fluorescence recovery after photobleaching suggested that the forming mineral conformed to the cellulosic cell wall, leaving the cytoplasm well connected to neighboring cells. As the silicified wall thickened, the functional cytoplasm shrunk into a very small space. These results imply that leaf silica deposition is an active, physiologically regulated process as opposed to a simple precipitation.

Most photorespiratory genes are preferentially expressed in the bundle sheath cells of the C4 grass Sorghum bicolor.

One of the hallmarks of C4 plants is the division of labor between two different photosynthetic cell types, the mesophyll and the bundle sheath cells. C4 plants are of polyphyletic origin and, during the evolution of C4 photosynthesis, the expression of thousands of genes was altered and many genes acquired a cell type-specific or preferential expression pattern. Several lines of evidence, including computational modeling and physiological and phylogenetic analyses, indicate that alterations in the expression of a key photorespiration-related gene, encoding the glycine decarboxylase P subunit, was an early and important step during C4 evolution. Restricting the expression of this gene to the bundle sheath led to the establishment of a photorespiratory CO2 pump. We were interested in whether the expression of genes related to photorespiration remains bundle sheath specific in a fully optimized C4 species. Therefore we analyzed the expression of photorespiratory and C4 cycle genes using RNA in situ hybridization and transcriptome analysis of isolated mesophyll and bundle sheath cells in the C4 grass Sorghum bicolor It turns out that the C4 metabolism of Sorghum is based solely on the NADP-dependent malic enzyme pathway. The majority of photorespiratory gene expression, with some important exceptions, is restricted to the bundle sheath.

Distribution, structure and biosynthetic gene families of (1,3;1,4)-ß-glucan in Sorghum bicolor.

In cereals, the presence of soluble polysaccharides including (1,3;1,4)-ß-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.) Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-ß-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by high performance liquid chromatography (HPLC) and ranged from 2.6-3:1 in the grain, while ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-ß-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-ß-glucan influenced by temporal and spatial factors. The sorghum orthologs of genes implicated in the synthesis of (1,3;1,4)-ß-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative polymerase chain reaction, indicating that, as in other cereals, CslF6 transcripts dominated.

Development and function of caryopsis transport tissues in maize, sorghum and wheat.

Cereal caryopsis transport tissues are essential channels via which nutrients are transported into the embryo and endosperm. There are differences and similarities between caryopsis transport tissues of maize, sorghum and wheat. Vascular bundle, endosperm transfer cells, endosperm conducting cells and embryo surrounding region are common in maize, sorghum and wheat. Placentochalaza is special in maize and sorghum, while chalaza and nucellar projection transfer cells are special in wheat. There is an obvious apoplastic cavity between maternal and filial tissues in sorghum and wheat caryopses, but there is no obvious apoplastic cavity in maize caryopsis. Based on the latest research, the development and function of the three cereal caryopsis transport tissues are discussed and investigated in this paper.

The changes of GA level and signaling are involved in the regulation of mesocotyl elongation during blue light mediated de-etiolation in Sorghum bicolor.

De-etiolation during seedling development is antagonistically regulated by blue light (BL) and gibberellins (GAs). The crosstalk between blue light (BL) and GA metabolism and signaling remains unclear. Using the mutant har1 which is specifically hypersensitive to BL in de-etiolation, the involvement possibility of the GA metabolism, GA signaling in the inhibition of mesocotyl elongation of the sorghum (Sorghum bicolor L. var. R111) seeding under BL was investigated. The inhibition of mesocotyl and cell elongation by BL was restored by application of exogenous GA(3) in har1. The endogenous GA(3) level correspondingly decreased in har1 mesocotyl especially from 1 to 4 h after BL irradiation. Putative genes of GA metabolism enzymes SbGA20ox, SbGA3ox and SbGA2ox were detected by Real-Time PCR and the results showed that one of the SbGA2ox homologs appeared significantly higher transcript level in har1 than in R111 at 2 h after BL irradiation. Putative homologous genes of DELLAs increased after BL irradiation and were higher in har1 among the three homologs. Remarkable increase of the DELLA expression was observed responding to exogenous paclobutrazol (PAC). Our research provided evidence in monocot sorghum, that the changes of a set of the GA metabolism and signaling genes might be involved in BL-induced inhibition of cell elongation.

Apospory appears to accelerate onset of meiosis and sexual embryo sac formation in sorghum ovules.

BACKGROUND: Genetically unreduced (2n) embryo sacs (ES) form in ovules of gametophytic apomicts, the 2n eggs of which develop into embryos parthenogenetically. In many apomicts, 2n ES form precociously during ovule development. Whether meiosis and sexual ES formation also occur precociously in facultative apomicts (capable of apomictic and sexual reproduction) has not been studied. We determined onset timing of meiosis and sexual ES formation for 569 Sorghum bicolor genotypes, many of which produced 2n ES facultatively. RESULTS: Genotype differences for onset timing of meiosis and sexual ES formation, relative to ovule development, were highly significant. A major source of variation in timing of sexual germline development was presence or absence of apomictic ES, which formed from nucellar cells (apospory) in some genotypes. Genotypes that produced these aposporous ES underwent meiosis and sexual ES formation precociously. Aposporous ES formation was most prevalent in subsp. verticilliflorum and in breeding lines of subsp. bicolor. It was uncommon in land races. CONCLUSIONS: The present study adds meiosis and sexual ES formation to floral induction, apomictic ES formation, and parthenogenesis as processes observed to occur precociously in apomictic plants. The temporally diverse nature of these events suggests that an epigenetic memory of the plants' apomixis status exists throughout its life cycle, which triggers, during multiple life cycle phases, temporally distinct processes that accelerate reproduction.

Nitrogen deficiency results in changes to cell wall composition of sorghum seedlings.

Sorghum [Sorghum bicolor (L.) Moench] has been gaining attention as a feedstock for biomass energy production. While it is obvious that nitrogen (N) supply significantly affects sorghum growth and biomass accumulation, our knowledge is still limited regarding the effect of N on the biomass quality of sorghum, such as the contents and structures of lignin and other cell wall components. Therefore, in this study, we investigated the effects of N supply on the structure and composition of sorghum cell walls. The cell walls of hydroponically cultured sorghum seedlings grown under sufficient or deficient N conditions were analyzed using chemical, two-dimensional nuclear magnetic resonance, gene expression, and immunohistochemical methods. We found that the level of N supply considerably affected the cell wall structure and composition of sorghum seedlings. Limitation of N led to a decrease in the syringyl/guaiacyl lignin unit ratio and an increase in the amount and alteration of tissue distribution of several hemicelluloses, including mixed linkage (1 → 3), (1 → 4)-ß-D-glucan, and arabinoxylan. At least some of these cell wall alterations could be associated with changes in gene expression. Nitrogen status is thus one of the factors affecting the cell wall properties of sorghum seedlings.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes.

BACKGROUND: Sugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at approximately 12x. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level. RESULTS: The microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons. CONCLUSIONS: The sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.

Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent.

In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate environment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes.

Adaptive defence-related changes in the metabolome of Sorghum bicolor cells in response to lipopolysaccharides of the pathogen Burkholderia andropogonis.

Plant cell suspension culture systems are valuable for the study of complex biological systems such as inducible defence responses and aspects of plant innate immunity. Perturbations to the cellular metabolome can be investigated using metabolomic approaches in order to reveal the underlying metabolic mechanism of cellular responses. Lipopolysaccharides from the sorghum pathogen, Burkholderia andropogonis (LPSB.a.), were purified, chemically characterised and structurally elucidated. The lipid A moiety consists of tetra- and penta-acylated 1,4'-bis-phosphorylated disaccharide backbone decorated by aminoarabinose residues, while the O-polysaccharide chain consists of linear trisaccharide repeating units of [→2)-α-Rha3CMe-(1 → 3)-α-Rha-(1 → 3)-α-Rha-(1 → ]. The effect of LPSB.a. in triggering metabolic reprogramming in Sorghum bicolor cells were investigated using untargeted metabolomics with liquid chromatography coupled to mass spectrometry detection. Cells were treated with LPSB.a. and the metabolic changes monitored over a 30 h time period. Alterations in the levels of phytohormones (jasmonates, zeatins, traumatic-, azelaic- and abscisic acid), which marked the onset of defence responses and accumulation of defence-related metabolites, were observed. Phenylpropanoids and indole alkaloids as well as oxylipins that included di- and trihydroxyoctadecedienoic acids were identified as signatory biomarkers, with marked secretion into the extracellular milieu. The study demonstrated that sorghum cells recognise LPSB.a. as a 'microbe-associated molecular pattern', perturbing normal cellular homeostasis. The molecular features of the altered metabolome were associated with phytohormone-responsive metabolomic reconfiguration of primary and secondary metabolites originating from various metabolic pathways, in support of defence and immunity.

Metabolon formation in dhurrin biosynthesis.

Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., Høj, P.B., Møller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging.

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.

Chromosome identification and nomenclature of Sorghum bicolor.

Linkage group identities and homologies were determined for metaphase chromosomes of Sorghum bicolor (2n = 20) by FISH of landed BACs. Relative lengths of chromosomes in FISH-karyotyped metaphase spreads of the elite inbred BTx623 were used to estimate the molecular size of each chromosome and to establish a size-based nomenclature for sorghum chromosomes (SBI-01-SBI-10) and linkage groups (LG-01 to LG-10). Lengths of arms were determined to orient linkage groups relative to a standard karyotypic layout (short arms at top). The size-based nomenclature for BTx623 represents a reasonable choice as the standard for a unified chromosome nomenclature for use by the sorghum research community.

Prospecting for Energy-Rich Renewable Raw Materials: Sorghum Stem Case Study.

Sorghum vegetative tissues are becoming increasingly important for biofuel production. The composition of sorghum stem tissues is influenced by genotype, environment and photoperiod sensitivity, and varies widely between varieties and also between different stem tissues (outer rind vs inner pith). Here, the amount of cellulose, (1,3;1,4)-ß-glucan, arabinose and xylose in the stems of twelve diverse sorghum varieties, including four photoperiod-sensitive varieties, was measured. At maturity, most photoperiod-insensitive lines had 1% w/w (1,3;1,4)-ß-glucan in stem pith tissue whilst photoperiod-sensitive varieties remained in a vegetative stage and accumulated up to 6% w/w (1,3;1,4)-ß-glucan in the same tissue. Three sorghum lines were chosen for further study: a cultivated grain variety (Sorghum bicolor BTx623), a sweet variety (S. bicolor Rio) and a photoperiod-sensitive wild line (S. bicolor ssp. verticilliflorum Arun). The Arun line accumulated 5.5% w/w (1,3;1,4)-ß-glucan and had higher SbCslF6 and SbCslH3 transcript levels in pith tissues than did photoperiod-insensitive varieties Rio and BTx623 (<1% w/w pith (1,3;1,4)-ß-glucan). To assess the digestibility of the three varieties, stem tissue was treated with either hydrolytic enzymes or dilute acid and the release of fermentable glucose was determined. Despite having the highest lignin content, Arun yielded significantly more glucose than the other varieties, and theoretical calculation of ethanol yields was 10 344 L ha-1 from this sorghum stem tissue. These data indicate that sorghum stem (1,3;1,4)-ß-glucan content may have a significant effect on digestibility and bioethanol yields. This information opens new avenues of research to generate sorghum lines optimised for biofuel production.

Host specificity in Sporisorium reilianum is determined by distinct mechanisms in maize and sorghum.

Smut fungi are biotrophic plant pathogens that exhibit a very narrow host range. The smut fungus Sporisorium reilianum exists in two host-adapted formae speciales: S. reilianum f. sp. reilianum (SRS), which causes head smut of sorghum, and S. reilianum f. sp. zeae (SRZ), which induces disease on maize. It is unknown why the two formae speciales cannot form spores on their respective non-favoured hosts. By fungal DNA quantification and fluorescence microscopy of stained plant samples, we followed the colonization behaviour of both SRS and SRZ on sorghum and maize. Both formae speciales were able to penetrate and multiply in the leaves of both hosts. In sorghum, the hyphae of SRS reached the apical meristems, whereas the hyphae of SRZ did not. SRZ strongly induced several defence responses in sorghum, such as the generation of H2 O2 , callose and phytoalexins, whereas the hyphae of SRS did not. In maize, both SRS and SRZ were able to spread through the plant to the apical meristem. Transcriptome analysis of colonized maize leaves revealed more genes induced by SRZ than by SRS, with many of them being involved in defence responses. Amongst the maize genes specifically induced by SRS were 11 pentatricopeptide repeat proteins. Together with the microscopic analysis, these data indicate that SRZ succumbs to plant defence after sorghum penetration, whereas SRS proliferates in a relatively undisturbed manner, but non-efficiently, on maize. This shows that host specificity is determined by distinct mechanisms in sorghum and maize.

Observation and investigation of three endosperm transport tissues in sorghum caryopses.

Endosperm transport tissues in sorghum caryopses include endosperm transfer cells, endosperm conducting cells, and the embryo surrounding region. To elucidate the structural changes of these tissues and their relationship with the caryopsis development, sorghum caryopses were analyzed at different days after pollination using light, fluorescence, and electron microscopy. The following results were obtained: post-phloem maternal tissues included the placentochalaza and the nucellar projection-like nucellus. Well-developed endosperm transfer cells exhibited very evident flange-type wall ingrowths. Very few wall ingrowths were present in the initially developed endosperm transfer cells when the level of sucrose from the initially developed vascular system was low. At the middle stage of caryopsis development, the level of sucrose from the well-developed vascular system was high. Endosperm transfer cells increased in both area and layer amount, and their wall ingrowths increased in both length and density. Later in caryopsis development, the level of sucrose from the degenerated vascular system was low and wall ingrowths distorted in the degenerated endosperm transfer cells. Endosperm conducting cells primarily occupied the most part of endosperm, but decreased gradually because the upper part transformed into the starchy endosperm and the lower part degenerated to give space to the embryo growth. Although the embryo surrounding region initially enveloped the small embryo, it rapidly degenerated and finally disappeared. Our data showed that (1) the caryopsis vascular system influenced the differentiation of endosperm transfer cells by controlling the sugar levels (2) and configuration of endosperm transport tissues were probably altered to favor the growth of filial tissues.