Deciphering and prediction of transcriptome dynamics under fluctuating field conditions.

Determining the drivers of gene expression patterns is more straightforward in laboratory conditions than in the complex fluctuating environments where organisms typically live. We gathered transcriptome data from the leaves of rice plants in a paddy field along with the corresponding meteorological data and used them to develop statistical models for the endogenous and external influences on gene expression. Our results indicate that the transcriptome dynamics are predominantly governed by endogenous diurnal rhythms, ambient temperature, plant age, and solar radiation. The data revealed diurnal gates for environmental stimuli to influence transcription and pointed to relative influences exerted by circadian and environmental factors on different metabolic genes. The model also generated predictions for the influence of changing temperatures on transcriptome dynamics. We anticipate that our models will help translate the knowledge amassed in laboratories to problems in agriculture and that our approach to deciphering the transcriptome fluctuations in complex environments will be applicable to other organisms.

Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

WUSCHEL-related homeobox family genes in rice control lateral root primordium size.

The development of a plastic root system is essential for stable crop production under variable environments. Rice plants have two types of lateral roots (LRs): S-type (short and thin) and L-type (long, thick, and capable of further branching). LR types are determined at the primordium stage, with a larger primordium size in L-types than S-types. Despite the importance of LR types for rice adaptability to variable water conditions, molecular mechanisms underlying the primordium size control of LRs are unknown. Here, we show that two WUSCHEL-related homeobox (WOX) genes have opposing roles in controlling LR primordium (LRP) size in rice. Root tip excision on seminal roots induced L-type LR formation with wider primordia formed from an early developmental stage. QHB/OsWOX5 was isolated as a causative gene of a mutant that is defective in S-type LR formation but produces more L-type LRs than wild-type (WT) plants following root tip excision. A transcriptome analysis revealed that OsWOX10 is highly up-regulated in L-type LRPs. OsWOX10 overexpression in LRPs increased the LR diameter in an expression-dependent manner. Conversely, the mutation in OsWOX10 decreased the L-type LR diameter under mild drought conditions. The qhb mutants had higher OsWOX10 expression than WT after root tip excision. A yeast one-hybrid assay revealed that the transcriptional repressive activity of QHB was lost in qhb mutants. An electrophoresis mobility shift assay revealed that OsWOX10 is a potential target of QHB. These data suggest that QHB represses LR diameter increase, repressing OsWOX10 Our findings could help improve root system plasticity under variable environments.

A leaf-emanated signal orchestrates grain size and number in response to maternal resources.

In plants, variations in seed size and number are outcomes of different reproductive strategies. Both traits are often environmentally influenced, suggesting that a mechanism exists to coordinate these phenotypes in response to available maternal resources. Yet, how maternal resources are sensed and influence seed size and number is largely unknown. Here, we report a mechanism that senses maternal resources and coordinates grain size and number in the wild rice Oryza rufipogon, a wild progenitor of Asian cultivated rice. We showed that FT-like 9 (FTL9) regulates both grain size and number and that maternal photosynthetic assimilates induce FTL9 expression in leaves to act as a long-range signal that increases grain number and reduces size. Our findings highlight a strategy that benefits wild plants to survive in a fluctuating environment. In this strategy, when maternal resources are sufficient, wild plants increase their offspring number while preventing an increase in offspring size by the action of FTL9, which helps expand their habitats. In addition, we found that a loss-of-function allele (ftl9) is prevalent among wild and cultivated populations, offering a new scenario in the history of rice domestication.

MiRiQ Database: A Platform for In Silico Rice Mutant Screening.

Genetic studies using mutant resources have significantly contributed to elucidating plant gene function. Massive mutant libraries sequenced by next-generation sequencing technology facilitate mutant identification and functional analysis of genes of interest. Here, we report the creation and release of an open-access database (https://miriq.agr.kyushu-u.ac.jp/index.php), called Mutation-induced Rice in Kyushu University (MiRiQ), designed for in silico mutant screening based on a whole-genome-sequenced mutant library. This database allows any user to easily find mutants of interest without laborious efforts such as large-scale screening by PCR. The initial version of the MiRiQ database (version 1.0) harbors a total of 1.6 million single-nucleotide variants (SNVs) and InDels of 721 M1 plants that were mutagenized by N-methyl-N-nitrosourea treatment of the rice cultivar Nipponbare (Oryza sativa ssp. japonica). The SNVs were distributed among 87% of all 35,630 annotated protein-coding genes of the Nipponbare genome and were predicted to induce missense and nonsense mutations. The MiRiQ database provides built-in tools, such as a search tool by keywords and JBrowse for mutation searches. Users can request mutant seeds in the M2 or M3 generations from a request form linked to this database. We believe that the availability of a wide range of gene mutations in this database will benefit the plant science community and breeders worldwide by accelerating functional genomic research and crop improvement.

Transcriptome Analysis of Rice Root Tips Reveals Auxin, Gibberellin and Ethylene Signaling Underlying Nutritropism.

Nutritropism is a positive tropism toward nutrients in plant roots. An NH4+ gradient is a nutritropic stimulus in rice (Oryza sativa L.). When rice roots are exposed to an NH4+ gradient generated around nutrient sources, root tips bend toward and coil around the sources. The molecular mechanisms are largely unknown. Here, we analyzed the transcriptomes of the inside and outside of bending root tips exhibiting nutritropism to reveal nutritropic signal transduction. Tissues facing the nutrient sources (inside) and away (outside) were separately collected by laser microdissection. Principal component analysis revealed distinct transcriptome patterns between the two tissues. Annotations of 153 differentially expressed genes implied that auxin, gibberellin and ethylene signaling were activated differentially between the sides of the root tips under nutritropism. Exogenous application of transport and/or biosynthesis inhibitors of these phytohormones largely inhibited the nutritropism. Thus, signaling and de novo biosynthesis of the three phytohormones are necessary for nutritropism. Expression patterns of IAA genes implied that auxins accumulated more in the inside tissues, meaning that ammonium stimulus is transduced to auxin signaling in nutritropism similar to gravity stimulus in gravitropism. SAUR and expansin genes, which are known to control cell wall modification and to promote cell elongation in shoot gravitropism, were highly expressed in the inside tissues rather than the outside tissues, and our transcriptome data are unexplainable for differential elongation in root nutritropism.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Neuroimaging of Neonatal Stroke: Venous Focus.

Perinatal venous infarcts are underrecognized clinically and at imaging. Neonates may be susceptible to venous infarcts because of hypercoagulable state, compressibility of the dural sinuses and superficial veins due to patent sutures, immature cerebral venous drainage pathways, and drastic physiologic changes of the brain circulation in the perinatal period. About 43% of cases of pediatric cerebral sinovenous thrombosis occur in the neonatal period. Venous infarcts can be recognized by ischemia or hemorrhage that does not respect an arterial territory. Knowledge of venous drainage pathways and territories can help radiologists recognize characteristic venous infarct patterns. Intraventricular hemorrhage in a term neonate with thalamocaudate hemorrhage should raise concern for internal cerebral vein thrombosis. A striato-hippocampal pattern of hemorrhage indicates basal vein of Rosenthal thrombosis. Choroid plexus hemorrhage may be due to obstruction of choroidal veins that drain the internal cerebral vein or basal vein of Rosenthal. Fan-shaped deep medullary venous congestion or thrombosis is due to impaired venous drainage into the subependymal veins, most commonly caused by germinal matrix hemorrhage in the premature infant and impeded flow in the deep venous system in the term infant. Subpial hemorrhage, an underrecognized hemorrhage stroke type, is often observed in the superficial temporal region, and its cause is probably multifactorial. The treatment of cerebral sinovenous thrombosis is anticoagulation, which should be considered even in the presence of intracranial hemorrhage. ©RSNA, 2024 Test Your Knowledge questions in the supplemental material and the slide presentation from the RSNA Annual Meeting are available for this article.

RASopathies for Radiologists.

RASopathies are a heterogeneous group of genetic syndromes caused by germline mutations in a group of genes that encode components or regulators of the Ras/mitogen-activated protein kinase (MAPK) signaling pathway. RASopathies include neurofibromatosis type 1, Legius syndrome, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, central conducting lymphatic anomaly, and capillary malformation-arteriovenous malformation syndrome. These disorders are grouped together as RASopathies based on our current understanding of the Ras/MAPK pathway. Abnormal activation of the Ras/MAPK pathway plays a major role in development of RASopathies. The individual disorders of RASopathies are rare, but collectively they are the most common genetic condition (one in 1000 newborns). Activation or dysregulation of the common Ras/MAPK pathway gives rise to overlapping clinical features of RASopathies, involving the cardiovascular, lymphatic, musculoskeletal, cutaneous, and central nervous systems. At the same time, there is much phenotypic variability in this group of disorders. Benign and malignant tumors are associated with certain disorders. Recently, many institutions have established multidisciplinary RASopathy clinics to address unique therapeutic challenges for patients with RASopathies. Medications developed for Ras/MAPK pathway-related cancer treatment may also control the clinical symptoms due to an abnormal Ras/MAPK pathway in RASopathies. Therefore, radiologists need to be aware of the concept of RASopathies to participate in multidisciplinary care. As with the clinical manifestations, imaging features of RASopathies are overlapping and at the same time diverse. As an introduction to the concept of RASopathies, the authors present major representative RASopathies, with emphasis on their imaging similarities and differences. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

Temporal changes in transcripts of miniature inverted-repeat transposable elements during rice endosperm development.

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs may escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. Genome-wide DNA demethylation is also observed in the endosperm after fertilization. In the present study, we used a custom microarray to survey the transcripts generated from TEs during rice endosperm development and at selected time points in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially those of miniature inverted-repeat TEs (MITEs). Some TE transcripts were directionally controlled, whereas the other DNA transposons and retrotransposons were not. We also discovered the NUCLEAR FACTOR Y binding motif, CCAAT, in the region near the 5' terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

Control of Plant Cell Growth and Proliferation by MO25A, a Conserved Major Component of the Mammalian Sterile 20-Like Kinase Pathway.

The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type-specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1, which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth.

Pediatric focal calvarial lesions: an illustrated review.

Focal skull lesions in children can be diagnostically challenging with a wide variety of potential etiologies. Understanding the diverse pathologies and recognizing their associated clinical and imaging characteristics is crucial for accurate diagnosis and appropriate treatment planning. We review pertinent anatomy of the scalp and calvarium and review different pathologies that can present with focal skull lesions in pediatric patients. These include neoplastic, non-neoplastic tumor-like, congenital, post traumatic, and vascular-associated etiologies. We review the key clinical and imaging features associated with these pathologies and present teaching points to help make the correct diagnosis. It is important for radiologists to be aware of the common and rare etiologies of skull lesions as well as the clinical and imaging characteristics which can be used to develop an accurate differential to ensure a timely diagnosis and initiate appropriate management.

Designing rice panicle architecture via developmental regulatory genes.

Rice panicle architecture displays remarkable diversity in branch number, branch length, and grain arrangement; however, much remains unknown about how such diversity in patterns is generated. Although several genes related to panicle branch number and panicle length have been identified, how panicle branch number and panicle length are coordinately regulated is unclear. Here, we show that panicle length and panicle branch number are independently regulated by the genes Prl5/OsGA20ox4, Pbl6/APO1, and Gn1a/OsCKX2. We produced near-isogenic lines (NILs) in the Koshihikari genetic background harboring the elite alleles for Prl5, regulating panicle rachis length; Pbl6, regulating primary branch length; and Gn1a, regulating panicle branching in various combinations. A pyramiding line carrying Prl5, Pbl6, and Gn1a showed increased panicle length and branching without any trade-off relationship between branch length or number. We successfully produced various arrangement patterns of grains by changing the combination of alleles at these three loci. Improvement of panicle architecture raised yield without associated negative effects on yield-related traits except for panicle number. Three-dimensional (3D) analyses by X-ray computed tomography (CT) of panicles revealed that differences in panicle architecture affect grain filling. Importantly, we determined that Prl5 improves grain filling without affecting grain number.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.

Transcriptome and hyperspectral profiling allows assessment of phosphorus nutrient status in rice under field conditions.

Phosphorus (P) is one of the macronutrients indispensable for crop production, and therefore it is important to understand the potential of plants to adapt to low P conditions. We compared growth and leaf genome-wide transcriptome of four rice cultivars during growth between two fields with different amount of available phosphate and further analysed the acceptable range of P levels for normal growth from the view of both appearance traits and internal P nutrient status, which was measured by profiling the expression of the P indicator gene. This demonstrated that rice plants have a robustness to moderate P-deficient conditions expressing a system for P acquisition and usage without any effects on yield potential and that P indicator gene expression could be a useful index for early diagnosis of P status in plants. To develop a simple method for assessment of P status, we tried to predict the expression level using reflectance spectroscopy and hyperspectral imaging, thereby providing models with good performance. Our findings suggest that rice plants have the potential to adapt to moderate low P conditions in the field and showed that the hyperspectral technique is one of the useful tools for simple measurement of molecular-level dynamics reflecting internal nutrient conditions.

Genome-wide analysis of spatiotemporal expression patterns during rice leaf development.

BACKGROUND: Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions. RESULTS: In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves. CONCLUSIONS: Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice.

BACKGROUND AND AIMS: Cold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy. METHODS: After exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities. KEY RESULTS: We observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress. CONCLUSIONS: The pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.

High-resolution spatiotemporal transcriptome analyses during cellularization of rice endosperm unveil the earliest gene regulation critical for aleurone and starchy endosperm cell fate specification.

The major tissues of the cereal endosperm are the starchy endosperm (SE) in the inner and the aleurone layer (AL) at the outer periphery. The fates of the cells that comprise these tissues are determined according to positional information; however, our understanding of the underlying molecular mechanisms remains limited. Here, we conducted a high-resolution spatiotemporal analysis of the rice endosperm transcriptome during early cellularization. In rice, endosperm cellularization proceeds in a concentric pattern from a primary alveolus cell layer, such that developmental progression can be defined by the number of cell layers. Using laser-capture microdissection to obtain precise tissue sections, transcriptomic changes were followed through five histologically defined stages of cellularization from the syncytial to 3-cell layer (3 L) stage. In addition, transcriptomes were compared between the inner and the outermost peripheral cell layers. Large differences in the transcriptomes between stages and between the inner and the peripheral cells were found. SE attributes were expressed at the alveolus-cell-layer stage but were preferentially activated in the inner cell layers that resulted from periclinal division of the alveolus cell layer. Similarly, AL attributes started to be expressed only after the 2 L stage and were localized to the outermost peripheral cell layer. These results indicate that the first periclinal division of the alveolus cell layer is asymmetric at the transcriptome level, and that the cell-fate-specifying positional cues and their perception system are already operating before the first periclinal division. Several genes related to epidermal identity (i.e., type IV homeodomain-leucine zipper genes and wax biosynthetic genes) were also found to be expressed at the syncytial stage, but their expression was localized to the outermost peripheral cell layer from the 2 L stage onward. We believe that our findings significantly enhance our knowledge of the mechanisms underlying cell fate specification in rice endosperm.